Neurodegenerative diseases and methods of modeling

ABSTRACT

Disclosed are embryonic stem cells and motor neurons derived from mice carrying transgenic alleles of the normal or mutant human SOD1 gene. Also disclosed are in vitro systems employing such SOD1 transgenic motor neurons for the study of neural degenerative disease.

RELATED APPLICATIONS

This application is copending with, shares at least one common inventorwith, and claims the benefit of U.S. Provisional Application No.61/124,229, filed Apr. 15, 2008, and U.S. Provisional Application No.61/200,293, filed Nov. 26, 2008. The entire contents of the priorapplications are hereby incorporated by reference.

GOVERNMENT SUPPORT

This invention was made with Government support under RO1 HD046732-01A1awarded by the National Institute of Health. The Government has certainrights in the invention.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

BACKGROUND

Amyotrophic Lateral Sclerosis (“ALS”), also known as Lou Gehrig'sdisease, is a progressive neurodegenerative disease characterized by theloss of upper and lower motor neurons, culminating in muscle wasting anddeath from respiratory failure (Boillee, S., Vande Velde, C. &Cleveland, D. W. ALS: a disease of motor neurons and their nonneuronalneighbors. Neuron 52, 39-59, 2006). The majority of ALS cases areapparently sporadic, with 90% of patients presenting disease symptomswithout a family history of ALS. The remaining 10% of ALS patients arediagnosed with familial ALS (Boillee et al., 1996; Brown, R. H., Jr.Amyotrophic lateral sclerosis. Insights from genetics. Arch Neurol 54,1246-50, 1997; Cole, N. & Siddique, T. Genetic disorders of motorneurons. Semin Neurol 19, 407-18, 1999). Approximately 25% of thefamilial cases of ALS are caused by dominant mutations in the geneencoding super oxide dismutase (SOD1) (Rosen, D. R. et al. Mutations inCu/Zn superoxide dismutase gene are associated with familial amyotrophiclateral sclerosis. Nature 362, 59-62, 1993). Identification ofpathogenic alleles of SOD1 has led to the production of transgenic mouseand rat models for the study of ALS (Gurney, M. E. et al. Motor neurondegeneration in mice that express a human Cu,Zn superoxide dismutasemutation. Science 264, 1772-5, 1994; Nagai, M. et al. Rats expressinghuman cytosolic copper-zinc superoxide dismutase transgenes withamyotrophic lateral sclerosis: associated mutations develop motor neurondisease. J Neurosci 21, 9246-54, 2001; Bruijn, L. I. et al. ALS-linkedSOD1 mutant G85R mediates damage to astrocytes and promotes rapidlyprogressive disease with SOD1-containing inclusions. Neuron 18, 327-38,1997; Wong, P. C. et al. An adverse property of a familial ALS-linkedSOD1 mutation causes motor neuron disease characterized by vacuolardegeneration of mitochondria. Neuron 14, 1105-16, 1995). Overproductionof pathogenic human SOD1 protein in mice and rats leads to late onset,progressive neurodegenerative disease (Gurney et al., 1994; Bruijn etal., 1997; Wong et al., 1995). Studies of the SOD1 animal models haveled to the identification and study of intrinsic pathogeniccharacteristics of ALS motor neurons including the formation of proteinaggregates, cytoskeletal abnormalities, proteosome dysfunction andincreased sensitivity to cell death signals (Boillee et al., 2006;Bruijn, L. I., Miller, T. M. & Cleveland, D. W. Unraveling themechanisms involved in motor neuron degeneration in ALS. Annu RevNeurosci 27, 723-49, 2004).

Studies of chimeric mice suggest that non-cell autonomous processescontribute to motor neuron death in ALS (Clement, A. M. et al. Wild-typenonneuronal cells extend survival of SOD1 mutant motor neurons in ALSmice. Science 302, 113-7, 2003). In animals bearing both wild-type cellsand cells harboring the SOD1G93A transgene, wild-type neurons surroundedby transgenic non-neuronal cells acquired cellular phenotypescharacteristic of ALS (Clement et al., 2003). Conversely, transgenicneurons associated with wild-type non-neuronal cells were increasinglyspared. However, these animal studies did not identify which cells wereinvolved in the pathological interactions with motor neurons due to thecomplex cellular milieu of both the spinal chord and the muscle.Conditional mutagenesis experiments in which the SOD1 transgene wasspecifically removed from motor neurons and microglial cells led to anincrease in animal lifespan, again suggesting the SOD1 protein can haveboth cell autonomous and non-cell autonomous affects in the disease(Boillee, S. et al. Onset and progression in inherited ALS determined bymotor neurons and microglia. Science 312, 1389-92, 2006). However, theseexperiments could not address the direct effect of cellular interactionswith motor neurons in the disease because of the use of death as anendpoint.

BRIEF SUMMARY OF THE INVENTION

In certain embodiments, the present invention provides an embryonic stemcell comprising a mutation in a gene involved in motor neurondevelopment and/or maintenance. In certain embodiments, the presentinvention provides a motor neuron generated by differentiating such anembryonic stem cell under conditions wherein the embryonic stem celladopts a motor neuron cell fate. In certain embodiments, the presentinvention provides an embryonic stem cell and/or a motor neuroncomprising a mutation in the SOD1 gene. For example, an embryonic stemcell and/or motor neuron of the present invention may comprise a SOD1mutation wherein a glycine is substituted for the wild type alanine atposition 93 of the SOD1 amino acid sequence (referred to herein as a“SOD1G93A” mutation or allele). In certain embodiments, such a mutationin a SOD1 gene is associated with a neurodegenerative disease.

In certain embodiments, an embryonic stem (ES) cell is derived from amouse bearing a transgene comprising a SOD1 allele, such as withoutlimitation, a SOD1G93A allele. In certain embodiments, such a mousebears a transgene comprising a human SOD1G93A allele. Such a transgenicmouse is known to recapitulate many pathologies of the human ALSdisease. In certain embodiments, an embryonic stem (ES) cell is a humanES cell bearing a transgene comprising a SOD1 allele, such as withoutlimitation, a SOD1G93A allele. In certain embodiments, transgenic EScells are differentiated into motor neurons in large numbers (e.g., suchas by one or more methods described in Wichterle, H., Lieberam, I.,Porter, J. A. & Jessell, T. M. Directed differentiation of embryonicstem cells into motor neurons. Cell 110, 385-97, 2002) and co-culturedeither with ES-derived cells that arise during the differentiationprocess and/or with other cells that contribute to the survival,maintenance and/or differentiation of such transgenic ES cells. Forexample, transgenic ES cells may be differentiated into motor neurons inthe presence of primary mouse and/or human glial cells. In certainembodiments, such primary mouse and/or human glial cells comprise awild-type genotype. In certain embodiments, such primary mouse and/orhuman glial cells comprise a non-wild-type genotype. For example, suchglial cells may comprise a mutant SOD1 allele, e.g., a SOD1G93A allele.Such a mutant SOD1 allele may be provided as a transgene.

In certain embodiments, motor neurons of such cultures display one ormore abnormalities typical of a phenotype observed in a particulardisease. In certain embodiments, motor neurons of such cultures displayone or more abnormalities typical of a phenotype observed in aneurodegenerative disease. For example, such motor neurons may displayabnormalities typical of those seen in the motor neurons of ALS patientsand/or ALS transgenic animals.

In certain embodiments, the present invention provides novel in vitromodel systems to study ALS and/or other neurodegenerative diseases, inwhich the factors directly influencing motor neuron development,differentiation and/or survival can be investigated. Certain of suchsystems are based on the differentiation of embryonic stem (ES) cellsderived from mice comprising a mutant SOD1 allele. Certain of suchsystems are based on the differentiation of human embryonic stem (ES)cells comprising a mutant SOD1 allele. An exemplary mutant SOD1 allelethat can be used in accordance with methods and compositions of thepresent invention is the SOD1G93A mutation, although systems of thepresent invention are not limited to this mutation.

In certain embodiments, in vitro model systems of the present inventionare used to screen for a test agent that affects the development,differentiation and/or survival of motor neurons. In certainembodiments, such in vitro model systems are used to screen for a testagent that affects the survival of wild type motor neurons. In certainembodiments, such in vitro model systems are used to screen for a testagent that affects the development, differentiation and/or survival ofmotor neurons that comprise one or more mutations. For example, suchmutant motor neurons may comprise a mutation associated with aneurodegenerative disease, such as for example, ALS. In certainembodiments, such mutant motor neurons comprise a mutation in the SOD1gene, e.g. a SOD1G93A mutation. In certain embodiments, the inventionprovides methods of identifying an agent that affects the survival of amutant motor neuron. For example, certain of such methods compriseproviding a mutant motor neuron comprising a SOD1 mutant allele,providing a test agent, contacting the mutant motor neuron with the testagent, and determining the effect of the test agent on survival of themutant motor neuron by comparing the survival of the mutant motor neuronto the survival of a control motor neuron lacking the SOD1 mutantallele, which control motor neuron is contacted with the test agent fora period of time and under conditions identical to that of the SOD1mutant motor neuron. In certain embodiments, a test agent is a cell, asmall molecule, a hormone, a vitamin, a nucleic acid molecule, anenzyme, an antibody, an amino acid, and/or a virus. In certainembodiments, the test agent is an agent that reduces the expression oractivity of a gene or a product of a gene in Table 2 (e.g., a product ofa gene in Table 2 which is involved in inflammation, an immune response,transcription, signaling, or a metabolic pathway). In certainembodiments, the test agent is an agent that reduces the expression oractivity of a prostaglandin D receptor.

In certain embodiments, the invention provides methods of identifying afactor that has a non-cell autonomous effect on the survival of a motorneuron. For example, certain of such methods comprise providing a motorneuron, identifying a first cell, which first cell negatively affectssurvival of the motor neuron, identifying a second cell, which secondcell does not negatively affect survival of the motor neuron, isolatinga factor from the either the first or second cell, wherein the factor iseither: i) a factor from the first cell that contributes to the negativeeffect on survival of the motor neuron; or ii) a factor from the secondcell that contributes to survival of the motor neuron. In certainembodiments, the first cell, second cell or both is a glial cell. Incertain embodiments, the first cell, second cell or both comprises amutation that is associated with amyloid lateral sclerosis, e.g. a SOD1mutation such as without limitation a SOD1G93A allele.

In certain embodiments, the invention provides methods of identifying afactor that has a non-cell autonomous effect on the survival of a motorneuron. For example, certain of such methods comprise providing a motorneuron, culturing the motor neuron in the presence of a test cell suchthat survival of the motor neuron is negatively affected as compared tosurvival of a motor neuron cultured in the presence of a control cell,and identifying a factor present in the test cell, which factorcontributes to the negative effect on survival of the motor neuron. Incertain embodiments, the invention provides methods of identifying afactor that has a non-cell autonomous effect on the survival of a motorneuron, which methods comprise providing a motor neuron, culturing themotor neuron in the presence of a test cell such that survival of themotor neuron is negatively positively affected as compared to survivalof a motor neuron cultured in the presence of a control cell, andidentifying a factor that is absent in the test cell, which factorcontributes to the positive effect on survival of the motor neuron.

In certain embodiments, the invention provides methods of identifying atest agent that modulates the non-cell autonomous effect of a test cellon the survival of a motor neuron. For example, certain of such methodscomprise providing a motor neuron, culturing the motor neuron in thepresence of a (i) test cell such that survival of the motor neuron isnegatively affected as compared to survival of a motor neuron culturedin the presence of a control cell, and (ii) a test agent, wherein achange in the survival of the motor neuron in the presence of the testagent as compared to the survival of the motor neuron in the absence ofthe test agent indicates that the test agent modulates the non-cellautonomous effect of a test cell. In certain embodiments, the test agentis an agent that reduces the expression or activity of a gene or aproduct of a gene in Table 2 (e.g., a product of a gene in Table 2 whichis involved in inflammation, an immune response, transcription,signaling, or a metabolic pathway). In certain embodiments, the testagent is an agent that reduces the expression or activity of aprostaglandin D receptor.

In certain embodiments, the invention provides methods of identifying afactor that has a cell autonomous effect on the survival of a motorneuron. For example, certain of such methods comprise providing a mutantmotor neuron comprising a first SOD1 mutation, providing a control motorneuron lacking the first SOD1 mutation, culturing the mutant motorneuron, determining the effect of the first SOD1 mutation on survival ofthe mutant motor neuron by comparing the survival of the mutant motorneuron to the survival of the control motor neuron, which control motorneuron is cultured for a period of time and under conditions identicalto that of the mutant motor neuron, and isolating a factor from theeither the mutant motor neuron or the control motor neuron, wherein thefactor is either: i) a factor from the mutant motor neuron thatcontributes to the negative effect on survival of the motor neuron; orii) a factor from the control motor neuron that contributes to survivalof the motor neuron.

In certain embodiments, in vitro model systems of the present inventionare used to screen for a factor that has a non-cell autonomous effect onthe development, differentiation and/or survival of a motor neuron, e.g.a SOD1 mutant motor neuron such as a SOD1G93A mutant motor neuron. Incertain embodiments, such a factor comprises a factor originating fromanother motor neuron. In certain embodiments, such a factor comprises afactor originating from another cell that is not a motor neuron. Incertain embodiments, such a factor originates from a glial cell. Such afactor may have a negative effect on the development, differentiationand/or survival of a motor neuron. Alternatively, such a factor maycontribute to the development, differentiation and/or survival of amotor neuron such that its absence negatively affects survival of themotor neuron.

In vitro model systems of the present invention are useful for theidentification of factors, agents, etc. that both positively andnegatively affect motor neuron survival. In certain embodiments, one ormore factors, agents, etc. that affect motor neuron survival areidentified by using SOD1 mutant glial cells, e.g. glial cells comprisinga SOD1G93A mutation. In certain embodiments, in vitro model systems ofthe present invention utilize human motor neurons and/or other celltypes derived from human ES cells. Such in vitro model systems may beadvantageously employed for the human physiological validation offindings from animal models, such as, without limitation, animal modelsthat recapitulate neurodegenerative diseases such as ALS and/or otherneurodegenerative diseases. Additionally or alternatively, such in vitromodel systems may be advantageously employed to identify novel factors,agents, etc. that affect human motor neuron development and/orcontribute to a disease state, such as, without limitation, aneurodegenerative disease, e.g. ALS, in the absence of an animal model.Additionally or alternatively, such in vitro model systems may beadvantageously employed to illuminate the target, efficacy, toxicity,mode of action, etc. of factors, agents, etc. that affect human motorneuron development and/or contribute to a disease state, such as,without limitation, a neurodegenerative disease, e.g. ALS.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows one embodiment of derivation of Hb9GFP; SOD1 mouse ES celllines. PCRs (FIG. 1A) for human SOD1 and Il2, and (FIG. 1B) for GFP inHb9::GFP, Hb9::GFP; SOD1 and Hb9GFP; SOD1G93A ES cell lines. (FIG. 1C)Expression of human SOD1 protein in Hb9GFP, Hb9GFP; SOD1 and Hb9GFP;SOD1G93A ES cell lines. (FIG. 1D) E10.5 chimera generated using theHb9GFP ES cell line.

FIG. 2 shows one embodiment of differentiation of the SOD1 G93A mouse EScell lines into motor neurons. (FIG. 2A) GFP expression of the ES celllines during different phases of the differentiation into motor neurons.Expression of neuronal markers (FIG. 2B) Tuj1, (FIG. 2C) Hb9, (FIG. 2D)Isl1, (FIG. 2E) Choline Acetyl-Transferase (ChAT) in motor neuronsderived from HB9GFP; SOD1G93A ES cell lines at 9 and 14 days after thebeginning of differentiation process. (FIG. 2F) Presence of GFAPpositive cells surrounding a GFP motor neuron after 28 days. The whitearrows indicate the nuclei of motor neurons immunoreactive for hb9 orIsl1 proteins.

FIG. 3 shows the effect of genetic background on motor neuron survival.(FIG. 3A) GFP positive motor neurons derived from SOD1G93A and Hb9GFP 61days after differentiation. Number of GFP positive cells derived from(FIG. 3B) Hb9GFP and (FIG. 3C) SOD1G93A ES cell lines present 15, 30, 45and 60 days after dissociation of EBs plated at two differentconcentrations (8×10⁵ and 4×10⁵ per well). (FIG. 3D) Number of GFPpositive motor neurons derived from Hb9GFP and SOD1G93A 15 and 30 daysafter EB dissociation plated at the concentration of 8×10⁵ (D) and 4×10⁵(F) per well. (FIGS. 3E, 3G). Same experiments in (FIGS. 3D, 3F)analyzed as percent of GFP positive motor neurons derived from Hb9GFPand SOD1G93A cell lines present at day 15, which still remain at 30days.

FIG. 4 shows intracellular aggregation of SOD1 protein in cultured motorneurons. Expression of human SOD1 protein in motor neurons derived from(FIG. 4A) SOD1G93A and (FIG. 4B) SOD1 cell lines at day 7, 14 and 21after EBs dissociation. (FIG. 4C) Co-expression of ubiquitin and SOD1protein in cells derived from the SOD1G93A cell line, 28 days afterdifferentiation. (FIG. 4D) Percentage of GFP-Positive motor neurons withSOD1 inclusions present after 21 days in culture.

FIG. 5 shows that glial cell genotype directly affects motor neuronsurvival in culture. (FIG. 5A) GFP-positive motor neurons at 5, 7, 14,21 and 28 days in culture after EB dissociation. (FIG. 5B) Graph showspercentage of Hb9GFP positive cells over time in all the conditionsstudied. Experiments were made in triplicate and results were normalizedto the number of cells found at 7 days in vitro.

FIG. 6 shows the percentage of differentiating EB cells that expressGFP. FACS analysis of cells dissociated from EBs after 5 days oftreatment with retinoic acid and shh. (FIG. 6A) Non transgenic cellline, (FIG. 6B) Hb9GFP, (FIG. 6C) Hb9GFP; SOD1, (FIG. 6D) Hb9GFP;SOD1G93A. The dot plots are representative of one experiment, but thepercentages are the average of three different experiments. Calcein bluewas used to assay the viability of cells during sorting.

FIG. 7 shows expression of neuronal marker Isl1 in cells derived fromSOD1G93A mouse ES cell lines. Expression of neuronal marker Isl1 in GFPpositive motor neurons (white arrows) and non-neuronal cells (whitestars) 14 days after differentiation.

FIG. 8 shows quantitative and qualitative analysis of SOD1 proteininclusions in ES cell derived motor neurons. (FIG. 8A) Average area ofSOD1 inclusions in SOD1 and SOD1G93A derived motor neurons. (FIG. 8B)Average length of SOD1 inclusions in SOD1 and SOD1G93A derived motorneurons. (FIG. 8C) Integrated Optical Density of SOD1 inclusions in SOD1and SOD1G93A derived motor neurons. (FIG. 8D) Representative SOD1G93A EScell derived motor neurons, 21 days after EB dissociation, displayingintracellular inclusion of the SOD1 protein. (FIG. 8E) Distribution ofinclusion bodies per cell in SOD1 and SOD1G93A derived motor neurons.Results are graphed as mean+/−S.E.M.

FIG. 9 shows activation of Caspase 3 and cytoplasmic localization ofCytochrome C in SOD1G93A motor neurons 14 days after EB dissociation.(FIG. 9A) A SOD1G93A derived motor neuron is immunoreactive with anantibody specific to the activated form of caspase 3 (white arrow).Other cell types are also immunoreactive for the staining (white stars).(FIG. 9B) The same motor neuron at a larger magnification is clearlyvisible in fragmented nuclei (DAPI) that is also immunoreactive for theantibody for Caspase 3. (FIG. 9C). The cytoplasm of a subset of motorneurons in culture also stained broadly with an antibody specific toCytochrome C, suggesting that cell death pathways are activated.

FIG. 10 shows characterization of primary glial monolayers derived fromSOD1 and SOD1 G93A mice. (FIG. 10A) Immuno-fluorescence Analysis of GFAPand S100 expression in SOD1 and SOD1G93A glial monolayers at 7 and 14days. (FIGS. 10B, 10C) Summary of immuno-fluorescent analysis of gliamarkers GFAP, 5100, RC2, Vimentin, CD 11 b, CNPase for both wt glia(FIG. 10B) and SOD1G93A glia (FIG. 10C) at different time points.

FIG. 11 shows one embodiment of differentiation of human ES cells intomotor neurons. (FIG. 11A) Diagram outlining the protocol used todifferentiate human ES cells into motor neurons: Undifferentiated humanES cell colonies are dissociated in collagenase, and grown as EBs forthe first 14 days in EB media, then are induced to a rostrocaudalidentity with retinoic acid (RA) and Shh for another 14 days. Finally,EBs are matured in the presence of GDNF for 14 more days. At this pointthe EBs can either be plated whole or dissociated with papain and thenplated. (FIG. 11B) Immunohistochemistry was performed to detect neuronalmarkers PAX 6, NKX 6.1, ISL1/2, and HB9 in EB sections at 14, 28, and 42days after collagenase treatment of undifferentiated human ES cells.(FIG. 11C) Percentage of cells immuno-reactive for HB9 after treatmentwith or without RA and Shh. (FIG. 11D) Percentage of cellsimmuno-reactive for HB9 after 42 days of differentiation in differentHuES cell lines.

FIG. 12 shows characterization of the Hb9::GFP human ES cell line. (FIG.12A) DNA construct used for the electroporation of human ES cells. (FIG.12B) GFP expression during different stages of the differentiation fromhuman ES cells into motor neurons. Expression of neuronal markers (FIG.12C) HB9, (FIG. 12D) PAX6, (FIG. 12E) ISL1/2, (FIG. 12F) CholineAcetyl-Transferase (ChAT) in motor neurons derived from Hb9::GFP humanES cells.

FIG. 13 shows the effect of glial cells over expressing SOD1G93A onhuman ES cell-derived motor neurons. (FIG. 13A) Experimental design:embryonic stem cells were differentiated into motor neurons, and anequal number of cells were plated on two different glial monolayers; onederived from mice over-expressing SOD1G93A, and the other derived fromnon-transgenic mice (WT). Motor neurons were counted after 10 and 20days in co-culture. (FIG. 13B) Number of HB9 positive cells 10 daysafter plating on SOD1G93A or non-transgenic (WT) glia. (FIG. 13C) Numberof HB9 positive cells 20 days after plating on SOD1G93A ornon-transgenic (WT) glia. (FIG. 13D) Images of HB9/Tuj1 positive cells20 days after plating on SOD1G93A glia or non-transgenic (WT) glia.(FIG. 13E) Number of Hb9::GFP cells 20 days after plating on SOD1G93Aglia or non-transgenic (WT) glia or glia over-expressing the wild typeform of human SOD1 (SOD1WT). Images of Hb9::GFP cells in the threedifferent co-culture conditions (FIGS. 13F-13H).

FIG. 14 shows the specificity of the toxic effect of glia overexpressingSOD1G93A on motor neurons. (FIG. 14A) Experimental design: embryonicstem cells were differentiated into motor neurons, and an equal numberof cells were plated on two different glial monolayers; one derived frommice over-expressing the mutation SOD1G93A, and the other derived fromnon-transgenic mice (WT). Human ES cell derived interneurons werecounted after 20 days in co-culture using two different markers, CHX10and LHX2. (FIG. 14B) Number of LHX2 positive cells 20 days after platingon SOD1G93A glia or non-transgenic (WT) glia. (FIG. 14C) Number of CHX10positive cells 20 days after plating on SOD1 G93A or non-transgenic (WT)glia. (FIG. 14D) Image of LHX2/Tuj1 positive cells 20 days after platingon SOD1G93A glia or non-transgenic (WT) glia. (FIG. 14E) Image ofCHX10/Tuj1 positive cells 20 days after plating on SOD1G93A glia ornon-transgenic (WT) glia. (FIG. 14F) Experimental design: embryonic stemcells were differentiated into motor neurons and same number of cellswas plated on two different MEF monolayers; one derived from miceover-expressing the mutation SOD1G93A, and the other derived fromnon-transgenic mice (WT). Motor neurons were counted after 20 days tocompare the two conditions. (FIG. 14G) Image of HB9/Tuj1 positive cells20 days after plating on SOD1G93A or non transgenic (WT) MEF. (FIG. 14H)Number of HB9 positive cells 20 days after plating on SOD1 G93A ornon-transgenic (WT) MEF.

FIG. 15 shows neuronal marker expression at different time points duringone embodiment of differentiation from human ES cells toward the motorneuron fate. (FIG. 15A) Percent of sectioned EBs (n=20) stainingpositive for PAX6, NKX6.1, ISL1/2, or HB9 at day 0, day 14, day 28, andday 42 of differentiation. (FIG. 15B) Percent of cells per sectioned EB(n=3) staining positive for PAX6, NKX6.1, ISL1/2, or HB9 at day 0, day14, day 28, and day 42 of differentiation.

FIG. 16 shows the effect of glia on human ES cell derived neurons. (FIG.16A) Expression of the glial marker glial fibrillary acidic protein(GFAP) and the neuronal marker Tuj1 in mouse-derived glia cells. (FIGS.16B-16C) Different density and morphology of human neurons 1 day afterplating on a glial layer or on laminin.

FIG. 17 shows analysis of one embodiment of human ES cell derived motorneurons. Expression of neuronal markers (FIG. 17A) ISL1/2, (FIG. 17B)HB9, (FIG. 17C) Choline Acetyl-Transferase (ChAT) in motor neuronsderived from human ES cells.

FIG. 18 shows characterization of a Hb9::GFP human ES cell line. (FIGS.18A-18C) Expression of HB9 protein in Hb9::GFP positive cells. (FIG.18D) Number of Hb9::GFP cells that are immunoreactive to Hb9 antibody(Hb9+Hb9::GFP), or not immunoreactive to Hb9 antibody (Hb9::GFP only).Expression of neuronal markers (FIG. 18E) NKX2.2, (FIG. 18F) NKX6.1,(FIG. 18G) LHX2, (FIG. 18H) CHX10, in motor neurons derived fromHb9::GFP human ES cells.

FIG. 19 shows one embodiment of overexpression of human SOD1G93A thatresults in protein aggregation in mouse ES cell derived motor neurons,but not glia. (FIG. 19A) Human SOD1 expression in mouse motor neuronsderived from Hb9::GFP mouse ES cells over expressing SOD1G93A, after 21days in culture. (FIG. 19B) Human SOD1 expression in glia derived frommice over-expressing the mutation SOD1G93A, after 3 months in culture.

FIG. 20A is a Venn Diagram presenting the overlap among transcriptsselectively over expressed in SOD1G93A glia and in SOD1 WT glia withrespect to WT glia.

FIG. 20B is a table listing a subset of genes over expressed in SOD1G93A glia but not in SOD1 WT glia or WT glia.

FIG. 20C is a graph showing the percentage of Hb9::GFP cells remainingon non-transgenic (WT) glia after 20 days of treatment with GMFb,Rantes, Cxcl 7, Mcp 2, Shh or PGD2 compared to the untreated condition(Ctrl) (n=3).

FIG. 20D shows images of Hb9::GFP positive cells after 20 days oftreatment with prostaglandin D2 (PGD2) or without treatment (Ctrl) on WTGlia.

FIG. 20E is a graph showing the percentage of Hb9::GFP cells remainingon WT glia or SOD1G93A glia after 20 days of treatment with theinhibitor of Prostaglandin D2 receptor (MK 0524) (n=3).

FIG. 20F shows images of Hb9::GFP positive cells after 20 days oftreatment with an inhibitor of Prostaglandin D2 receptor (MK 0524) orwithout treatment (Ctrl) on SOD1G93A glia.

DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

In certain embodiments, the present invention provides compositions andmethods for detailed mechanistic studies of the interactions betweencells such as, without limitation, motor neurons and other cells suchas, without limitation, glia. In certain embodiments, a motor neuron ofthe present invention comprises a mutant motor neuron comprising anallele associates with a neurodegenerative disease, such as, withoutlimitation, ALS. In certain embodiments, a mutant motor neuron comprisesa SOD1 mutant allele associated with ALS. For example, a mutant motorneuron may comprise a SOD1G93A allele. In certain embodiments,compositions and methods of the present invention provide an assay fordiffusible factor(s), agent(s), etc. toxic to motor neurons. In certainembodiments, the present invention provides a high throughput cell basedassay for small molecules that promote survival of mutant SOD1 motorneurons. The present disclosure validates the use of ES cells carryingdisease-causing genes to study disease mechanisms.

Certain embodiments of the present invention are discussed in detailbelow. Those of ordinary skill in the art will understand, however, thatvarious modifications to these embodiments are within the scope of theappended claims. It is the claims and equivalents thereof that definethe scope of the present invention, which is not and should not belimited to or by this description of certain embodiments.

DEFINITIONS

“Agent”, “Test agent”: The terms “agent” and “test agent” as used hereinrefer to a compound or other entity that is tested to determine whetherit has an effect on the differentiation, development and/or survival ofa cell. As non-limiting examples, a test agent may comprise a cell, asmall molecule, a hormone, a vitamin, a nucleic acid molecule, anenzyme, an amino acid, and/or a virus. Those of ordinary skill in theart will be aware of other test agents that may be tested for theireffect(s) on differentiation, development and/or survival of a cell. Incertain embodiments, a differentiating cell is subjected to a test agentbefore, during and/or after differentiation to determine its effect(s)on differentiation, development and/or survival of a cell. For example,an embryonic stem cell undergoing differentiation into a cell type ofinterest may be subjected to a test agent before, during and/or afterdifferentiation. In certain embodiments, an embryonic stem cellundergoing differentiation into a motor neuron is subjected to a testagent before, during and/or after differentiation. In certainembodiments, a test agent that is identified as having one or moreeffects on the differentiation, development and/or survival of a cell isused in the treatment, prevention and/or cure of a disease of interest.

“Embryonic stem cell”, “ES cell”: The terms “embryonic stem cell” and“ES cell” as used herein refer to an undifferentiated stem cell that isderived from the inner cell mass of a blastocyst embryo and ispluripotent, thus possessing the capability of developing into any organor tissue type or, at least potentially, into a complete embryo.Embryonic stem cells appear to be capable of proliferating indefinitely,and of differentiating into all of the specialized cell types of amammal, including the three embryonic germ layers (endoderm, mesoderm,and ectoderm), and all somatic cell lineages and the germ line. Asnon-limiting examples, embryonic stem cells have been shown to becapable of being induced to differentiate into cardiomyocytes (Paquin etal., Proc. Nat. Acad. Sci., 99:9550-9555, 2002), hematopoietic cells(Weiss et al., Hematol. Oncol. Clin. N. Amer., 11(6):1185-98, 1997; alsoU.S. Pat. No. 6,280,718), insulin-secreting beta cells (Assady et al.,Diabetes, 50(8):1691-1697, 2001), and neural progenitors capable ofdifferentiating into astrocytes, oligodendrocytes, and mature neurons(Reubinoff et al., Nature Biotechnology, 19:1134-1140, 2001; also U.S.Pat. No. 5,851,832). One of ordinary skill in the art will be aware ofother cell types that have been derived from embryonic stem cells.

“SOD1”: As will be clear from context, the term “SOD1” as used hereinrefers to either the gene encoding superoxide dismutase 1 or the enzymeencoded by this gene. The SOD1 gene or gene product is known by othernames in the art including, but not limited to, ALS1, Cu/Zn superoxidedismutase, indophenoloxidase A, IPOA, and SODC_HUMAN. Those of ordinaryskill in the art will be aware of other synonymous names that refer tothe SOD1 gene or gene product. The SOD1 enzyme neutralizes superchargedoxygen molecules (called superoxide radicals), which can damage cells iftheir levels are not controlled. The human SOD1 gene maps to cytogeneticlocation 21q22.1. Certain mutations in SOD1 are associated with ALS inhumans including, but not limited to, Ala4Val, Gly37Arg and Gly93Ala,and more than one hundred others. Those of ordinary skill in the artwill be aware of these and other human mutations associated with ALS.Certain compositions and methods of the present invention comprise oremploy cells comprising a SOD1 mutation.

“Stem-cell producing condition”: The term “stem-cell producingcondition” as used herein refers to a condition or set of conditionsthat permits and/or drives a cell to become a stem cell. In certainembodiments, an embryonic cell is permitted and/or driven to become anembryonic stem cell by subjecting such an embryonic cell to a stem-cellproducing condition. For example, an embryonic blastomere may permittedand/or driven to become an embryonic stem cell by isolating theembryonic blastomere from the inner cell mass of a blastocyst andculturing the embryonic blastomere under stem-cell producing conditions,such that at least one blastomere proliferates into a pluripotentembryonic stem cell. In certain embodiments, a transgenic embryonic stemcell is generated by producing a transgenic cell according to one ormore methods of the present invention, allowing the transgenic cell todevelop into a transgenic blastocyst comprising a plurality oftransgenic blastomeres, isolating one or more transgenic blastomeresfrom the inner cell mass of the transgenic blastocyst, and culturing theisolated transgenic blastomere(s) under stem-cell producing conditionssuch that at least one transgenic blastomere develops into a pluripotenttransgenic embryonic stem cell.

Embryonic Stem Cells and their Generation

Stem cells typically share two important characteristics thatdistinguish them from other types of cells. First, they areunspecialized cells that are capable of maintaining their unspecializedstate and of renewing themselves for long periods through cell division.Second, under appropriate conditions, they can be induced todifferentiate into cells with specialized functions. Several types ofstem cells have been identified including adult stem cells, umbilicalcord stem cells, and embryonic stem cells.

Embryonic stem cells may be characterized by any of several criteria,which criteria will be known by those of ordinary skill in the art. Forexample, embryonic stem cells are typically capable of continuousindefinite replication in vitro. Continued proliferation for a longperiod of time (e.g., 6 months, one year or longer) of culture is asufficient evidence for immortality, as primary cell cultures withoutthis property fail to continuously divide for such a length of time(Freshney, Culture of animal cells. New York: Wiley-Liss, 1994). Incertain embodiments, embryonic stem will continue to proliferate invitro under appropriate culture conditions for longer than one year, andmaintain the developmental potential to contribute all three embryonicgerm layers throughout this time. Such developmental potential can bedemonstrated by the injection of embryonic stem cells that have beencultured for a prolonged period (over a year) into SCID mice and thenhistologically examining the resulting tumors. However, length of timein culture is not the sole criteria that may be used to identify anembryonic stem cell, and even though cells have grown in culture forless than 6 months, such cells may nevertheless be embryonic stem cells.

Additionally or alternatively, embryonic stem cells may be identified bythe expression of certain markers, including but not limited to cellsurface markers. As will be understood by those of ordinary skill in theart, embryonic stem cells from different species will exhibitspecies-specific markers on their cell surfaces. For example, Thomson(U.S. Pat. Nos. 5,843,780 and 6,200,806, each of which is incorporatedherein in its entirety by reference) discloses certain cell surfacemarkers that may be used to identify embryonic stem cells derived fromprimates. Furthermore, Stage Specific Embryonic Antigens (SSEAs) aremonoclonal antibodies that recognize defined carbohydrate epitopes andmay also be used to identify embryonic stem cells. Embryonic stem cellsderived from different species exhibit different patterns of SSEAs. Forexample, undifferentiated primate ES cells (including human ES cells)express SSEA-3 and SSEA-4, but not SSEA-1. Conversely, undifferentiatedmouse ES cells express SSEA-1, but not SSEA-3 or SSEA-4. Additionally oralternatively, markers that are not exhibited on the surface of a cellmay be used to identify an embryonic stem cell. For example, thehomeodomain transcription factor Oct 4 (also termed Oct-3 or Oct3/4) isfrequently used as a marker for totipotent embryonic stem cells. Thoseof ordinary skill in the art will be aware of cell surface and othermarkers that are useful in identifying embryonic stem cells, includingmarkers diagnostic of a given species, that can be used to identify anembryonic stem cell from that species.

Additionally or alternatively, embryonic stem cells may be identified bythe capacity to develop into all of the specialized cell types of amammal, including the three embryonic germ layers (endoderm, mesoderm,and ectoderm), and all somatic cell lineages and the germ line.Additionally and/or alternatively, embryonic stem cells may beidentified by the capacity to participate in normal development whentransplanted into a preimplantation embryo to generate a chimericembryo.

Cultured cells that have proliferated in cell culture for a long periodof time (e.g., six or more months) without differentiating, arepluripotent, and appear genetically normal are typically considered tobe embryonic stem cells. In certain embodiments, an embryonic stem cellof the present invention comprises a human embryonic stem cell. Incertain embodiments, an embryonic stem cell of the present inventioncomprises a non-human embryonic stem cell. For example, a non-humanembryonic stem cell of the present invention may include, but is notlimited to, a mouse, rat, pig, sheep, goat, and/or a primate stem cell.Those of ordinary skill in the art will be aware of other non-human stemcells that may be used in accordance with the present invention.

The capacity of embryonic stem cells (ES) to self renew in culture,while retaining their pluripotent potential, provides the opportunity toproduce virtually unlimited numbers of differentiated cell types toreplenish those lost as a consequence of disease (Evans, M. J. &Kaufman, M. H. Establishment in culture of pluripotential cells frommouse embryos. Nature 292, 154-6, 1981; Martin, G. R. Isolation of apluripotent cell line from early mouse embryos cultured in mediumconditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 78,7634-8, 1981). An alternative, but equally important potential of EScells is to provide insights into disease mechanisms (Lerou, P. H. &Daley, G. Q. Therapeutic potential of embryonic stem cells. Blood Rev19, 321-31, 2005; Ben-Nun, I. F. & Benvenisty, N. Human embryonic stemcells as a cellular model for human disorders. Mol Cell Endocrinol 252,154-9, 2006). ES cells carrying the genes responsible for a particulardisease can be induced to differentiate into the cell types affected inthat disease. Studies of the differentiated cells in culture couldprovide important information regarding the molecular and cellularnature of events leading to pathology.

In certain embodiments, this approach is used to develop an in vitromodel of Amyotrophic Lateral Sclerosis (“ALS”). As described more fullybelow in the Examples section of the present application, embryonic stemcell lines were derived from normal mice, and from mice thatover-express either the wild-type human SOD1 transgene or the mutantSOD1G93A transgene, the latter of which is responsible for one type offamilial ALS (see Example section). Using the methods established byWichterle et al (2002) the three ES cell lines were differentiated intomotor neurons in culture. The wild-type SOD1 and the mutant SOD1G93Amotor neurons produce high levels of the corresponding human SOD1proteins, and they both display properties that characterize bone fidemotor neurons. These motor neurons could be maintained in long-termculture, providing the opportunity to detect differences between themutant SOD1G93A ES cell-derived motor neurons and those derived fromcontrol cell lines.

In certain embodiments, embryonic stem cells are generated by culturingcells from the inner cell mass in a culture dish that is coated with afeeder layer comprising mouse embryonic skin cells that have beentreated so they will not divide. Such a feeder layer gives the innercell mass cells a sticky surface to which they can attach and alsoreleases nutrients into the culture medium. In certain embodiments,cells from the inner cell mass are cultured in a culture dish that isnot coated with a feeder layer. Such embodiments provide certainadvantages including reduction of the risk that viruses or othermacromolecules in the mouse cells may be transmitted to the culturedcells.

In certain embodiments, embryonic stem cells are generated by subjectingcells to stem-cell producing conditions. Stem-cell producing conditionsare known to those of ordinary skill in the art and can often varybetween species. For example, leukemia inhibitory factor (LIF) isnecessary and sufficient to prevent differentiation of mouse embryonicstem cells and to allow them to grow in an undifferentiated stateindefinitely. Conversely, for primate embryonic stem cells, at least onegroup has reported that growth on a fibroblast feeder layer is requiredto prevent them from differentiating (see e.g., U.S. Pat. Nos. 5,843,780and 6,200,806, incorporated herein by reference in their entirety). Oneof ordinary skill in the art will be aware of appropriate stem-cellproducing conditions including, but not limited to, culture media and/orculturing conditions that permit and/or drive a cell of a given speciesto become a stem cell.

In certain embodiments, embryonic stem cells of the present inventionare generated by any of a variety of methods disclosed in U.S.Provisional Patent Application No. 60/926,525, filed Apr. 26, 2007,which is incorporated herein by reference in its entirety. For example,in certain embodiments, an embryonic stem cell is generated bytransferring nuclear-derived genetic material from a donor cell to arecipient cell to generate a transgenic cell, after which the transgeniccell is allowed to develop into a blastocyst and a blastomere cell fromthe inner cell mass is isolated and/or cultured (and optionally passagedfor several generations) under stem-cell producing conditions, resultingin generation of an embryonic stem cell syngenic with thenuclear-derived genetic material removed from the donor cell used togenerate the transgenic cell.

In certain embodiments, embryonic stem cells of the present inventionare generated such that the generated embryonic stem cells comprise amutation associated with a disease of interest. For example, embryonicstem cells may be generated which contain a mutation associated with aneurodegenerative disease. Exemplary neurodegenerative diseases include,but are not limited to: ALS, Parkinson's disease, and Alzheimer'sdisease. Those of ordinary skill in the art will be aware of otherneurodegenerative diseases of interest, as well as mutations associatedwith such diseases.

In certain embodiments, an embryonic stem cell is generated thatcomprises a mutation associated with ALS. For example, an embryonic stemcell may be generated that comprises a mutation in the SOD1 gene, e.g.,Ala4Val, Gly37Arg and/or Gly93Ala. In certain embodiments, an embryonicstem cell is generated that comprises a SOD1G93A allele. In certainembodiments, an embryonic stem cell is generated that comprises a humanSOD1G93A allele.

In certain embodiments, an embryonic stem cell comprises a mutation in agene associated with neurodegenerative disease, which gene is present asa transgene. For example, an endogenous gene associated with aneurodegenerative disease may be deleted or otherwise inactivated insuch an embryonic stem cell by any of a variety of techniques known tothose skilled in the art, and a transgene comprising a mutant copy ofthe endogenous gene may be introduced into the embryonic stem cell byany of a variety of techniques known to those skilled in the art. Incertain embodiments, such a transgene is integrated into the genome ofthe embryonic stem cell. In certain embodiments, such a transgene is notintegrated into the genome of the embryonic stem cell.

Once embryonic stem cell lines are established, batches of suchembryonic stem cell lines can be frozen and for future culturing and/orexperimentation.

Differentiation into Motor Neurons

In certain embodiments of the present invention, an embryonic stem cellis subjected to conditions that result in the embryonic stem celldifferentiating into a motor neuron. For example, embryonic stem cellsmay be dissociated into a single-cell suspension, allowed tospontaneously aggregate into embryoid bodies over a first period of time(e.g. 48 hours, although such a period of time may be increased ordecreased depending on other conditions to which the embryonic stemcells are subjected), and then treated with a suitable differentiationfactor or factors for a second period of time such that the embryonicstem cells differentiate into motor neurons. By way of example, suchdifferentiation factors may include retinoic acid (RAc) and solublesonic hedgehog (Shh), which may be administered for, e.g., 5 days. Otherdifferentiation factor(s) and condition(s) will be known to those ofordinary skill in the art.

In certain embodiments, a motor neuron differentiated from an embryonicstem cell comprises a mutation in a gene associated withneurodegenerative disease. As non-limiting examples, such aneurodegenerative disease may include ALS, Parkinson's disease,Alzheimer's disease or any number of other neurodegenerative diseasesknown to those of skill in the art. A variety of genes are known to beassociated with neurodegenerative diseases. As one non-limiting example,mutations in the SOD1 gene are known to be associated with theneurodegenerative disease ALS. For example, in humans, Gly92Ala, Ala4Valand Gly37Arg mutations are associated with the onset and progression ofALS. Those of ordinary skill in the art will be aware of other SOD1mutations associated with ALS. In certain embodiments, compositions andmethod of the present invention comprise or employ human motor neuronscomprising a SOD1G93A mutation such as Gly92Ala, Ala4Val and/orGly37Arg.

In mice, the dominant SOD1G93A mutation is associated with ALS-likephenotype. Thus, in certain embodiments, the present invention comprisesmouse motor neurons comprising a SOD1G93A mutation. In certainembodiments, the present invention comprises human motor neuronscomprising a SOD1G93A mutation.

A number of changes characteristic of neurodegeneration in ALS wereobserved in mouse mutant SOD1G93A motor neurons between 14 and 28 days(for additional detail, see Examples section below). First, the SOD1G93Aprotein changed its intracellular localization, forming inclusions thatincreased in size and density. Second, the levels of ubiquitinincreased. Third, some motor neurons expressed activated caspase-3 anddisplayed cytoplasmic staining with cytochrome c antibodies. Finally, asignificant difference in survival was observed between mutant SOD1G93Amotor neurons and the controls. Thus, many of the late onset pathologiesobserved in both human ALS and SOD1G93A mice are recapitulated in thisin vitro model, including the loss of motor neurons, which is ultimatecause of symptoms in patients.

In certain embodiments, methods of the present invention comprise usinghuman and/or non-human SOD1 mutant motor neurons to screen for testagents that affect motor neuron differentiation, development and/orsurvival. In certain embodiments, methods of the present inventioncomprise using such SOD1 mutant motor neurons to identify a factor thathas a non-cell autonomous effect on the differentiation, developmentand/or survival of a motor neuron.

In certain embodiments, a motor neuron differentiated from an embryonicstem cell comprises a mutation in a gene associated withneurodegenerative disease, which gene is present as a transgene. Forexample, an endogenous gene associated with a neurodegenerative diseasemay be deleted or otherwise inactivated in an embryonic stem cell fromwhich such a motor neuron is derived by any of a variety of techniquesknown to those skilled in the art, and a transgene comprising a mutantcopy of the endogenous gene may be introduced into the embryonic stemcell by any of a variety of techniques known to those skilled in theart. In certain embodiments, such a transgene is integrated into thegenome of the differentiated motor neuron. In certain embodiments, sucha transgene is not integrated into the genome of the differentiatedmotor neuron.

Conditions Affecting Motor Neuron Differentiation, Development and/orSurvival

The present invention encompasses the recognition that properdifferentiation, development and/or survival of a cell can be influencedby its environment. For example, non-cell autonomous processes cancontribute to the differentiation, development and/or survival of acell. In certain embodiments, the present invention provides novelsystem and compositions for studying such non-cell autonomous processesand for identifying factors that mediate such non-cell autonomousprocesses.

In certain embodiments, methods and compositions of the presentinvention are used to study non-cell autonomous processes thatcontribute to the proper differentiation, development and/or survival ofa motor neuron. For example, both autonomous defects in motor neuronsand toxic non-cell autonomous interactions with other cell types in thespinal cord have been implicated in ALS pathology (Bruijn et al., 2004;Clement et al., 2003; Boillee et al., 1995; Beers, D. R. et al.Wild-type microglia extend survival in PU.1 knockout mice with familialamyotrophic lateral sclerosis. Proc Natl Acad Sci USA 103, 16021-6,2006). Methods and compositions of the present invention are well suitedto the identification and study of factors that mediate non-cellautonomous effects of other cell types on motor neurons, leading to ALS.

Several studies have suggested that cells within the spinal cord mayhave pathological, non-cell autonomous affects on motor neurons or onthe rate of disease progression (Clement et al., 2003; Boillee et al.,2006). However, these studies were of limited utility since they werenot able to resolve the identity of cell types that caused these affectsand/or were not able to determine whether they acted directly to affectmotor neuron survival. The present invention encompasses the discoveryand recognition that cultures of ES cell derived motor neurons containother cell types, including astroglia, and that these ES cell derivedcells have a non-cell autonomous affect on motor neuron survival invitro (see Examples section below). The effects of co-culturing motorneurons with primary glia from SOD1G93A mice and mice expressing thewild-type SOD1 protein were systematically examined. It was discoveredthat mutant SOD1G93A glia reduced the survival of both wild type andmutant motor neurons. However, the effect was significantly greater onmutant SOD1G93A motor neurons. Therefore, the presently describedstudies show for the first time that an ALS genotype in glial cellsdirectly and negatively affects the survival of motor neurons and theyconfirm that there is a cell autonomous component to motor neurondegeneration.

Consistent with the present disclosure, Nagai et al. have shown thatprimary astroctye cultures expressing ALS-associated mutant SOD1proteins contain diffusible factor(s) that are toxic to both primary andES cell-derived motor neurons (Makiko Nagai, D. B. R., Tetsuya Nagata,Alcmene Chalazonitis, Thomas M. Jessell, Hynek Wichterle, SergePrzedborski. Astrocytes expressing ALS-associated SOD1 mutants releasefactors selectively toxic to spinal motor neurons. Nature Neuroscience,2007). In Nagai et al.'s study, motor neurons were the only cell typesaffected by these mutant glial cells and only SOD1G93A glial cells, notmuscle cells or fibroblasts, adversely affected motor neuron survival.Although in Nagai et al's study, mutant primary neurons exhibitedmorphometric alterations, their survival up to 14 days in culture wasindistinguishable from that of their wild-type counterparts. In thepresently described studies, differences in survival between wild-typeSOD1 and mutant SOD1G93A ES cell-derived motor neurons were observed at14 and 28 days in culture. The differences between the two studies mayoriginate in the source (embryo or ES cell-derived) or number of themotor neurons used and the timeframe of the investigations.

In certain embodiments, the present invention provides methods foridentifying and studying non-cell autonomous factors produced by glialand/or other cells, which factors influence the differentiation,development and/or survival of motor neurons. For example, motor neuronsmay be cultured in the present of mutant glial cells, and the survivalof such motor neurons may be compared to the survival of motor neuronscultured in the presence of wild type glial cells. A difference insurvival of motor neurons indicates that a mutation present in such aglial cell is important in mediating proper survival of motor neurons.In certain embodiments, such a mutation in a glial cell results in analteration in the quantity and/or quality of a protein encoded by a genein which the mutation is located, which protein may be a factor thatcontributes to proper survival of motor neurons. In certain embodiments,a mutation in a glial cell results in an alteration in the quantityand/or quality of a protein that is not encoded by gene in which themutation is located. For example, a mutation may alter the quantityand/or quality of a produced transcription factor, which transcriptionfactor contributes to the proper regulation and/or expression of asecond protein, which second protein may be a factor that mediatesproper survival of motor neurons. In certain embodiments, a mutation ina glial cell results in an alteration in the quantity and/or quality ofa factor that contributes to proper survival of motor neurons, whichfactor is not a protein (e.g. a small molecule, a lipid, a hormone,etc.). Those of ordinary skill in the art will be aware of a variety ofother ways in which a mutation in a particular gene may affect a factorthat contributes to the proper survival of motor neurons.

In certain embodiments, embryonic stem cells are induced todifferentiate into motor neurons in the presence of glial cells. Suchembodiments are useful in the study of normal motor neurondifferentiation, development and/or survival, and can be expected toprovide useful insights into possible causes, treatments and/or cures ofvarious neurodegenerative diseases.

In certain embodiments, embryonic stem cells are induced todifferentiate into motor neurons in the presence of mutant glial cells,and differentiation, development and/or survival of such motor neuronsmay be compared to differentiation, development and/or survival of motorneurons cultured in the presence of wild type glial cells. A differencein differentiation, development and/or survival of the motor neuronsindicates that a mutation present in the glial cell contributes toproper differentiation, development and/or survival of motor neurons. Incertain embodiments, a mutation in a glial cell results in an alterationin the quantity and/or quality of a protein encoded by gene in which themutation is located, which protein is a factor that contributes toproper differentiation, development and/or survival of motor neurons. Incertain embodiments, a mutation in a glial cell results in an alterationin the quantity and/or quality of a protein that is not encoded by genein which the mutation is located. For example, a mutation may alter thequantity and/or quality of a produced transcription factor, whichtranscription contributes to the proper regulation and/or expression ofa second protein, which second protein is a factor that mediates properdifferentiation, development and/or survival of motor neurons. Incertain embodiments, a mutation in a glial cell results in an alterationin the quantity and/or quality of a factor that is important for properdifferentiation, development and/or survival of motor neurons, whichfactor is not a protein (e.g. a small molecule, a lipid, a hormone,etc.). Those of ordinary skill in the art will be aware of a variety ofother ways in which a mutation in a particular gene may affect a factorthat is important in the proper survival of motor neurons.

In certain embodiments, mutant glial cells to be used in accordance withthe present invention to identify and/or study factors that contributeto proper differentiation, development and/or survival of motor neuronscomprise a mutation in a gene associated with a neurodegenerativedisease. As but a few non-limiting examples, such a neurodegenerativedisease may include ALS, Parkinson's disease, Alzheimer's disease or anynumber of other neurodegenerative diseases known to those of skill inthe art. A variety of genes are known to be associated withneurodegenerative diseases. As one non-limiting example, mutations inthe SOD1 gene are known to be associated with the neurodegenerativedisease ALS. Thus, in certain embodiments, mutant glial cells to be usedin accordance with the present invention to identify and/or studyfactors that contribute to proper differentiation, development and/orsurvival of motor neurons comprise a mutation in the SOD1 gene. Inhumans, Gly92Ala, Ala4Val and Gly37Arg mutations are associated with theonset and progression of ALS. Thus, in certain embodiments, mutant glialcells to be used in accordance with the present invention to identifyand/or study factors that contribute to proper differentiation,development and/or survival of motor neurons comprise a SOD1G93Amutation such as Gly92Ala, Ala4Val and/or Gly37Arg. Those of ordinaryskill in the art will be aware of a variety of other SOD1 mutant allelesassociated with ALS, which mutant alleles can be advantageously used inaccordance with one or more of the embodiments described herein.

In certain embodiments, the present invention provides methods foridentifying and/or studying non-cell autonomous factors produced bynon-glial cells, which factors influence the differentiation,development and/or survival of motor neurons. Non-limiting examples ofsuch non-glial cells that can influence differentiation, developmentand/or survival of motor neurons include microglial cells,oligodendrocytes, astrocytes, other neuronal cell types in spinal cords(e.g. interneurons) and/or other cells that are in contact with neurons(e.g. muscle cells). Those of ordinary skill in the art will be aware ofa variety of other non-glial cells that can influence thedifferentiation, development and/or survival of motor neurons.

In certain embodiments, methods of the present invention compriseidentifying a factor that has a non-cell autonomous effect on survivalof a motor neuron. In certain embodiments, such methods compriseproviding a motor neuron, identifying a first glial cell, which firstglial cell negatively affects survival of the motor neuron, identifyinga second glial cell, which second glial cell does not negatively affectsurvival of the motor neuron, isolating a factor from the either thefirst or second glial cell, wherein the factor is either: i) a factorfrom the first glial cell that contributes to the negative effect onsurvival of the motor neuron; or ii) a factor from the second glial cellthat contributes to survival of the motor neuron.

Factors that influence differentiation, development and/or survival canbe identified by any of a variety of methods known to those of ordinaryskill in the art. In certain embodiments, such a factor is directlyidentified after determining that a given wild type or mutant cellcontributes to proper differentiation, development and/or survival of acell of interest such as, e.g., a motor neuron. Non-limiting examples ofsuch methods include fractionation, mass spectrometry, protein chipanalysis (e.g., if such a factor comprises a protein), chromatography,etc. Those of ordinary skill in the art will be aware of and will beable to employ suitable techniques for directly identifying such afactor.

In certain embodiments, such a factor is identified indirectly. Forexample, a factor may comprise a protein encoded by a gene. In suchembodiments, a gene that encodes such a factor may be identified by anyof a variety of techniques such as, for example, differential display,gene chip analysis, RT-PCR, direct sequencing, etc. Those of ordinaryskill in the art will be aware of and will be able to employ suitabletechniques for identifying such a factor indirectly.

In certain embodiments, a combination of two or more factors maytogether contribute to differentiation, development and/or survival of acell of interest such as, for example, a motor neuron. Methods andcompositions of the present invention may be advantageously used toidentify such a combination of factors.

Disease Modeling and Drug Screening

In certain embodiments, the present invention offers great potential fordeveloping better models for the study of human disease and/or bettermethods of treatment. In certain embodiments, methods of the presentinvention employ embryonic stem cells and/or differentiated cells thatcomprise alterations (e.g., deletions, rearrangements, duplications,substitutions, etc.) in genes associated with a particular disease. Incertain embodiments, a disease of interest is a neurodegenerativedisease. Exemplary neurodegenerative diseases that can be studied usingcompositions and methods of the present invention include, but are notlimited to, ALS, Parkinson's disease, and Alzheimer's disease. Thoseskilled in the art will be aware of a number of other neurodegenerativediseases that can be studied using compositions and methods the presentinvention.

In certain embodiments, a disease of interest is modeled and/or studiedby inducing an embryonic stem cell line (that has, for example, beengenerated by any of the variety of methods of the present invention tocontain one or more alterations in one or more genes associated with adisease of interest) to differentiate by culturing such a cell lineunder appropriate differentiation conditions. For example, an embryonicstem cell line may be generated that contains one or more alterations inone or more genes associated with a neurological degenerative disease,e.g. ALS, or any other neurodegenerative disease of interest. Such anembryonic cell line may be induced to differentiate into motor neuronsby subjecting it to appropriate differentiation conditions. Those ofordinary skill in the art will be aware of appropriate differentiationconditions. By observing differentiation, development and/or survival ofsuch a motor neuron and comparing it to differentiation, developmentand/or survival of a motor neuron derived from an embryonic stem cellline that does not contain the genetic alteration(s) associated with theneurodegenerative disease of interest, the practitioner can achieve abetter understanding of the genetic basis of disease progression andpathogenesis.

Additionally or alternatively, an embryonic stem cell line that isgenerated to contain one or more alterations in one or more genesassociated with a disease of interest may be used to screen for agents(e.g., a cell, a small molecule, a hormone, a vitamin, a nucleic acidmolecule, an enzyme, an antibody, an amino acid, a virus, etc.) that canbe used, for example, in the treatment, prevention and/or cure of thatdisease. For example, such an embryonic stem cell line may be induced todifferentiate into a cell type associated with the disease of interestby placing it under appropriate differentiation conditions. Before,during and/or after differentiation, such a cell may be subjected to atest agent in order to determine whether that agent has an effect ondifferentiation, development and/or survival of the cell. In certainembodiments, an embryonic stem cell comprising a mutation associatedwith a neurodegenerative disease is induced to differentiate into amotor neuron, which motor neuron is subjected to an agent before, duringand/or after differentiation.

In certain embodiments, compositions and methods of the presentinvention are useful in studying and/or modeling diseases that to date,have not been amenable to such study and/or modeling. For instance, inmany cases, by the time a patient is diagnosed with a particulardisease, the early events of disease progression and pathogenesis havealready occurred, making it difficult or impossible to determine andtrack the molecular, cellular, or other changes that occur during thecourse of the disease. Using inventive methods and compositionsdisclosed herein, researchers will now be able to determine and studysuch molecular, cellular, or other changes, leading to a betterunderstanding of disease progression and pointing the way to moreeffective treatments.

In certain embodiments, methods of the present invention compriseidentifying an agent that affects the survival of a SOD1 mutant motorneuron. In certain embodiments, such methods comprise providing a SOD1mutant motor neuron, providing a test agent, contacting the SOD1 mutantmotor neuron with the test agent, and determining the effect of the testagent on survival of the SOD1 mutant motor neuron by comparing thesurvival of the SOD1 mutant motor neuron to the survival of a controlmotor neuron lacking the SOD1 mutant allele, which control motor neuronis contacted with the test agent for a period of time and underconditions identical to that of the SOD1 mutant motor neuron. In certainembodiments, a SOD1 motor neuron used in such methods is derived from anembryonic stem cell. In certain embodiments, a SOD1 motor neuron used insuch methods comprises a SOD1 mutation associated with aneurodegenerative disease of interest, e.g. ALS.

In certain embodiments, compositions and methods of the presentinvention are used to study and/or model diseases and/or to screen foragents that can be used in the treatment, prevention and/or cure ofdiseases, which compositions or methods comprise or make use of humanembryonic stem cell lines and/or differentiated cells derived from suchhuman embryonic stem cell lines.

In certain embodiments, compositions and methods of the presentinvention are used to study and/or model diseases and/or to screen foragents that can be used in the treatment, prevention and/or cure ofdiseases, which compositions or methods comprise or make use ofnon-human embryonic stem cell lines and/or differentiated cells derivedfrom such non-human embryonic stem cell lines. Use of non-human stemcell lines and/or differentiated cells derived from them is advantageouswhen ethical and/or practical limitations prevent the use of human stemcell lines. Non-limiting examples of non-human stem cell lines (and/ordifferentiated cells derived from them) that may be used in accordancewith the present invention to study and/or model diseases and/or toscreen for agents that can be used in the treatment, prevention and/orcure of diseases include mouse, rat, and primate stem cell lines. Thoseof ordinary skill in the art will be aware of a variety of othernon-human stem cell lines that will be useful, and those of ordinaryskill in the art will be able to generate such stem cell lines byemploying one or more methods of the present invention.

Compositions and methods of the present invention may be used to studyand/or model of any of a variety of diseases or conditions. Non-limitingexamples of such diseases or conditions include childhood congenitalmalformations, sickle cell anemia, neurological diseases such asamyotrophic lateral sclerosis (also known as Lou Gehrig's disease),Parkinson's disease, Alzheimer's disease or any of a variety of otherneurological diseases, Down syndrome (a condition that arises inpatients with trisomy for chromosome 21 resulting in dysregulatedsignaling through the NFAT/calcineurin pathway), etc. One of ordinaryskill in the art will be aware of a variety of other disease conditionsthat may be modeled and/or studied by generating embryonic stem cellsaccording to one or more methods of the present invention.

In certain embodiments, compositions and methods of the presentinvention comprise or make use of human embryonic stem cell lines and/ordifferentiated cells derived from a patient suffering from and/orpredicted to suffer from a disease of interest. Patient-specific,immune-matched human embryonic stem cells have the potential to be ofgreat biomedical importance for studies of disease and development. Forexample, certain patients may respond better to a given therapy or drugregimen than other patients. Additionally or alternatively, certainpatients may experience fewer and/or less severe side effects afterbeing administered a given therapy or drug regimen than other patients.By utilizing embryonic stem cells that contain the genetic complement ofa patient suffering from and/or predicted to suffer from a disease ofinterest, and permitting such cells to differentiate into a cell typeassociated with that disease, it will be possible to better predictwhich therapy or drug regimen will be most beneficial and/or result inthe least detrimental side effects.

Furthermore, patient-specific human embryonic stem cells have thepotential to be of great biomedical importance for the discovery and/ordevelopment of patient-specific agents that can be used to preventand/or treat a disease of interest. By utilizing embryonic stem cellscontaining the genetic complement of a patient suffering from a diseaseof interest, permitting such cells to differentiate into a cell typeassociated with that disease, and subjecting such cells to one or moretest agents before, during and/or after differentiation, discoveryand/or development of an agent that will be most beneficial and/orresult in the least detrimental side effects for that particular patientwill be facilitated. Those of ordinary skill in the art will be able toapply methods and compositions of the present invention to the discoveryand/or development of an agent specific for a patient and/or disease ofinterest.

In certain embodiments, a disease of interest is modeled and/or studiedby inducing an embryonic stem cell line to differentiate into a celltype of interest by culturing such a cell line under appropriatedifferentiation conditions, wherein the embryonic stem cell line isdifferentiated in the presence of one or more different cell types thatcontribute to proper differentiation, development and/or survival of thecell type of interest. For example, an embryonic stem cell linecontaining one or more alterations in one or more genes associated witha neurological degenerative disease, e.g. ALS or any otherneurodegenerative disease of interest, may be induced to differentiateinto a motor neuron in the presence of one or more glial cells. Byobserving the differentiation, development and/or survival of such amotor neuron and comparing it to the differentiation, development and/orsurvival of a motor neuron derived from an embryonic stem cell line thatdoes not contain the genetic alteration(s) associated with theneurodegenerative disease of interest, the practitioner can achieve abetter understanding of the genetic basis of disease progression andpathogenesis.

In certain embodiments, an embryonic stem cell line containing a wildtype genome is induced to differentiate into a cell type of interest inthe presence of one or more different cell types that contribute toproper differentiation, development and/or survival of the cell type ofinterest. By observing the differentiation, development and/or survivalof such a cell type of interest and comparing it to the differentiation,development and/or survival of a cell type of interest in the absence ofsuch different cell types, the practitioner can achieve a betterunderstanding of the genetic basis of normal differentiation,development and/or survival a cell type of interest.

In certain embodiments, a cell type of interest is induced todifferentiate in the presence of one or more different cell types thatcontribute to proper differentiation, development and/or survival of thecell type of interest, wherein the one or more different cell typescomprise one or more mutations that affect differentiation, developmentand/or survival of the cell type of interest. By observing thedifferentiation, development and/or survival of the cell type ofinterest in such an environment and comparing it to the differentiation,development and/or survival of a cell type of interest differentiated inthe presence of wild type different cell types, the practitioner canachieve a better understanding of non-cell autonomous factors andprocesses that contribute to proper differentiation, development and/orsurvival of the cell type of interest.

In certain embodiments, the present invention provides systems andmethods for identifying agents (e.g., a cell, a small molecule, ahormone, a vitamin, a nucleic acid molecule, an enzyme, an antibody, anamino acid, a virus, etc.) that can be used, for example, in thetreatment, prevention and/or cure of a disease of interest, wherein thedifferentiation, development and/or survival of a cell type of interestimplicated in the onset and/or progression of the disease of interest isinfluenced by one or more different cell types. For example, such anembryonic stem cell line may be induced to differentiate into a celltype associated with the disease of interest by placing it underappropriate differentiation conditions in the presence of one or moredifferent cell types that contribute to proper differentiation,development and/or survival of the cell type of interest. Before, duringand/or after differentiation, such a cell may be subjected to a testagent in order to determine whether that agent has an effect ondifferentiation, development and/or survival of the cell.

In certain embodiments, an embryonic stem cell comprising a mutationassociated with a neurodegenerative disease is induced to differentiateinto a motor neuron in the presence of glial cells, which motor neuronis subjected to an agent before, during and/or after differentiation. Incertain embodiments, such glial cells are wild type. In certainembodiments, such glial cells comprise one or more mutations that alterthe proper differentiation, development and/or survival of the motorneuron. For example, such glial cells may comprise a mutation thatinduces the motor neuron to display a phenotype characteristic of adisease of interest, such as for example, a neurodegenerative diseaseincluding, without limitation, ALS. In certain embodiments, such glialcells comprise a mutation in the SOD1 gene, for example a SOD1G93Amutation. Thus, in certain embodiments, the present invention providessystems and methods for identifying agent(s) that prevent, ameliorate,or reverse the adverse effects of such SOD1G93A mutant glial cells onthe proper differentiation, development and/or survival of a motorneuron, including both wild type and mutant motor neurons. In certainembodiments, such identified agents are used to prevent, treat and/orcure ALS.

As described in the Examples herein, it has been discovered that certaingenes are overexpressed in glia having a mutation in the SOD1 gene.Moreover, it has been discovered that agents that target such genes orexpression products of such genes can promote survival of motor neurons(e.g., motor neurons produced and/or cultured according to a methoddescribed herein). Accordingly, in some embodiments, the presentinvention provides methods of identifying a test agent that modulatessurvival of a motor neuron, wherein the test agent targets a gene orproduct of a gene overexpressed in SOD1 mutant glia. In someembodiments, a test agent targets (e.g., inhibits expression or activityof) a gene or product of a gene in Table 2 (e.g., a gene or product of agene selected from serine (or cysteine) preptidase inhibitor, Glade A,member 1b (Serpina1b); protein tyrosine phosphatase, non-receptor type 7(Ptpn7); poly (ADP-ribose) polymerase family, member 12 (Zc3hdc1);prostaglandin D2 receptor (Ptgdr); glia maturation factor, beta (Gmfb);ATP-binding cassette, sub-family A (ABC1), member 5 (Abca5); developingbrain homeobox 2 (Dbx2); RAB6B, member RAS oncogene family (Rab6b);cut-like 1 (Cut11); adenosine deaminase (Ada); receptor coactivator 6interacting protein (Ncoa6ip); interferon-induced protein 35 (i1i35);RAB, member of RAS oncogene family-like 2A (Rab12a); STEAP family member4 (A1481214); cytoglobin (cygb); Duffy blood group, chemokine receptor(Dfy); chondrolectin (Chod1); neurexin 1 (NRXN1); defensin beta 11(Defb11); RUN and SH3 domain containing 2 (RUSC2); matrilin-4 (matn4);X-linked lymphocyte-regulated 3A (Xlr3a); C-C motif chemokine 8 (ccl8);T-cell immunoglobulin and mucin domain containing 4 (Timd4); odd-skippedrelated 2 (osr2); RIKEN cDNA 9130213B05 gene (9130213B05Rik);reversion-inducing-cysteine-rich protein with kazal motifs (Reck);olfactory receptor 116 (Olfr116); protogenin homolog (Prtg;A230098A12Rik); sonic hedgehog (Shh); formyl peptide receptor 1 (Fpr1);pro-platelet basic protein (chemokine (C-X-C motif) ligand 7; CXCL7);DnaJ (Hsp40) homolog, subfamily B, member 3 (DNAJB3); defensin beta 10(Deft 10); apolipoprotein A-II (Apoa2); collagen, type I, alpha 2(Col1a2); islet cell autoantigen 1-like (Ical1; 1700030B17Rik); ATPase,class II, type 9A (Atp9a); chemokine (C-C motif) ligand 5 (CCL5); solutecarrier family 39 (zinc transporter), member 14 (Sc139A14); serumamyloid A 3 (Saa3); RIKEN cDNA 3632451O06 gene (3632451O06Rik);attractin like 1 (Atrnl1); Alstrom syndrome 1 (Alms 1); NK2 homeobox 2(Nkx2-2); kallikrein related-peptidase 8 (Klk8; Prss19); histone cluster1, H4k (Hist1h4k); EPH receptor B2 (Ephb2); synaptotagmin XII (Syt12);forkhead box Q1 (Foxq1); splicing factor, arginine/serine-rich 16(Sfrs16); LanC lantibiotic synthetase component C-like 1 (Lancl1); andMARCKS-like 1 (M1p)). In some embodiments, a test inhibits expression oractivity of a gene or gene product in Table 2 which is involved ininflammation. In some embodiments, a test agent inhibits expression oractivity of a prostaglandin D receptor.

In certain embodiments, a test agent that inhibits expression oractivity of a gene or product of a gene in Table 2 includes a smallmolecule, an antibody, a hormone, a vitamin, a nucleic acid molecule, anenzyme, an amino acid, and/or a virus.

In certain embodiments, a test agent is a nucleic acid molecule, e.g., anucleic acid molecule that mediates RNA interference. RNA interferencerefers to sequence-specific inhibition of gene expression and/orreduction in target RNA levels mediated by an at least partlydouble-stranded RNA, which RNA comprises a portion that is substantiallycomplementary to a target RNA. Typically, at least part of thesubstantially complementary portion is within the double stranded regionof the RNA. In some embodiments, RNAi can occur via selectiveintracellular degradation of RNA. In some embodiments, RNAi can occur bytranslational repression. In some embodiments, RNAi agents mediateinhibition of gene expression by causing degradation of targettranscripts. In some embodiments, RNAi agents mediate inhibition of geneexpression by inhibiting translation of target transcripts. Generally,an RNAi agent includes a portion that is substantially complementary toa target RNA. In some embodiments, RNAi agents are at least partlydouble-stranded. In some embodiments, RNAi agents are single-stranded.In some embodiments, exemplary RNAi agents can include small interferingRNA (siRNA), short hairpin RNA (shRNA), and/or microRNA (miRNA). In someembodiments, an agent that mediates RNAi includes a blunt-ended (i.e.,without overhangs) dsRNA that can act as a Dicer substrate. For example,such an RNAi agent may comprise a blunt-ended dsRNA which is >25 basepairs length. RNAi mechanisms and the structure of various RNA moleculesknown to mediate RNAi, e.g. siRNA, shRNA, miRNA and their precursors,are described, e.g., in Dykxhhorn et al., 2003, Nat. Rev. Mol. Cell.Biol., 4:457; Hannon and Rossi, 2004, Nature, 431:3761; and Meister andTuschl, 2004, Nature, 431:343; all of which are incorporated herein byreference.

In some embodiments, a nucleic acid that mediates RNAi includes a 19nucleotide double-stranded portion, comprising a guide strand and anantisense strand. Each strand has a 2 nt 3′ overhang. Typically theguide strand of the siRNA is perfectly complementary to its target geneand mRNA transcript over at least 17-19 contiguous nucleotides, andtypically the two strands of the siRNA are perfectly complementary toeach other over the duplex portion. However, as will be appreciated byone of ordinary skill in the art, perfect complementarity is notrequired. Instead, one or more mismatches in the duplex formed by theguide strand and the target mRNA is often tolerated, particularly atcertain positions, without reducing the silencing activity below usefullevels. For example, there may be 1, 2, 3, or even more mismatchesbetween the target mRNA and the guide strand (disregarding theoverhangs).

Molecules having the appropriate structure and degree of complementarityto a target gene will exhibit a range of different silencingefficiencies. A variety of additional design criteria have beendeveloped to assist in the selection of effective siRNA sequences.Numerous software programs that can be used to choose siRNA sequencesthat are predicted to be particularly effective to silence a target geneof choice are available (see, e.g., Yuan et al., 2004, Nuc. Acid. Res.,32:W130; and Santoyo et al., 2005, Bioinformatics, 21:1376; both ofwhich are incorporated herein by reference).

As will be appreciated by one of ordinary skill in the art, RNAi may beeffectively mediated by RNA molecules having a variety of structuresthat differ in one or more respects from that described above. Forexample, the length of the duplex can be varied (e.g., from about 17-29nucleotides); the overhangs need not be present and, if present, theirlength and the identity of the nucleotides in the overhangs can vary(though most commonly symmetric dTdT overhangs are employed in syntheticsiRNAs).

In certain embodiments, the present invention provides methods ofmodulating survival of a motor neuron by contacting a motor neuron (invitro or in vivo) with an agent that reduces the expression or activityof a prostaglandin D receptor. Agents that reduce expression or activity(e.g., antagonize) prostaglandin D receptors include MK 0524((3R)-4-(4-chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]-aceticacid; Sturino et al., J. Med. Chem. 50(4):794-806, 2007) and analogsthereof, and compounds disclosed in Mitsumori et al., Curr. Pharm. Des.,10(28):3533-8, 2004; Beaulieu et al., Bioorg Med Chem Lett.18(8):2696-700, 2008; Torisu et al., Eur J Med Chem. 40(5):505-19, 2005;U.S. Pat. Pub. Nos. 20010051624, 20030055077, 20040180934, 20070244131,20070265278, 20070265291, 20080194600, and U.S. Pat. No. 7,153,852.

Human Stem Cells and Cells Derived from Them

Although mouse genetics has provided a sophisticated understanding ofthe cellular and molecular mechanisms that contribute to familial ALS,it cannot inform us as to the actual relevance of its findings to humanpatients. In fact, due to the fundamental differences between human andmouse physiology, many observations made in mouse disease models havenot translated well to human experimental systems or to the clinic. Forexample, diabetes has been “cured” many times over in the NOD mousemodel of disease. However, few of the observations and experimentaltherapies developed in this mouse model have proven relevant to thehuman disease (Shoda, L. K. et al. A comprehensive review ofinterventions in the NOD mouse and implications for translation.Immunity. 23, 115-26, 2005). Similarly, mutations in the RB gene thatlead to Retinoblastoma in human patients cause an independent range oftumors in mice carrying the same genetic lesion (Goodrich, D. W. Lee,W.H. Molecular characterization of the retinoblastoma susceptibilitygene. Biochim Biophys Acta. 1155, 43-61, 1993; Williams B. O. et al.Extensive contribution of Rb-deficient cells to adult chimeric mice withlimited histopathological consequences. EMBO J., 13, 4251-9, 1994). As aresult many therapeutics developed in animal, or based on drug-targetsdiscovered in animal models, fail in clinical trials (Shoda et al.,2005; Gawarylewski, A. The trouble with animal models. The Scientist. 21(7), 45-51, 2007; Rubin, L. L. Stem cells and drug discovery: thebeginning of a new era? Cell. 132, 549-52, 2008). The cost of thesefailures is substantial (Gawarylewski et al., 2007; Rubin, 2008).

Considerable time, effort and expense would be saved if fundamentalobservations made in animal models could be routinely validated in therelevant human cell types. A potential solution is to use humanembryonic stem cells as a renewable source of these cells for the studyof disease and for drug target validation. The discoveries describedherein have demonstrated the usefulness of motor neurons derived fromhuman ES cells in validating findings from mouse models of ALS (seee.g., Examples 7 and 8 below). It has been found that glia cellsover-expressing the SOD1G93A mutation negatively affect the viability ofhuman ES cell derived motor neurons in a time dependent manner. Suchnon-cell autonomous effect of glia is specific for motor neurons, as itdoes not seems interfere with the survival of human interneurons.

In certain embodiments, in vitro model systems of the present inventionutilize human motor neurons derived from human ES cells. In certainembodiments, a human motor neuron differentiated from a human embryonicstem cell comprises a mutation in a gene associated withneurodegenerative disease. As non-limiting examples, such aneurodegenerative disease may include ALS, Parkinson's disease,Alzheimer's disease or any number of other neurodegenerative diseasesknown to those of skill in the art. A variety of genes are known to beassociated with neurodegenerative diseases. As one non-limiting example,mutations in the SOD1 gene are known to be associated with theneurodegenerative disease ALS. For example, in humans, Gly92Ala, Ala4Valand Gly37Arg mutations are associated with the onset and progression ofALS. Those of ordinary skill in the art will be aware of other SOD1mutations associated with ALS. In certain embodiments, compositions andmethod of the present invention comprise or employ human motor neuronscomprising a SOD1G93A mutation such as Gly92Ala, Ala4Val and/orGly37Arg.

In certain embodiments, in vitro model systems of the present inventioncomprising human motor neurons may be advantageously employed for thehuman physiological validation of findings from animal models, such as,without limitation, animal models (e.g., mouse) that recapitulate inwhole or in part neurodegenerative diseases such as ALS and/or otherneurodegenerative diseases.

In certain embodiments, in vitro model systems of the present inventioncomprising human motor neurons may be advantageously employed toidentify novel factors, agents, etc. that affect motor neurondevelopment and/or contribute to a disease state, such as, withoutlimitation, a neurodegenerative disease, e.g. ALS, in the absence of ananimal model. In certain embodiments, in vitro model systems of thepresent invention comprising human motor neurons may be advantageouslyemployed to illuminate the target, efficacy, toxicity, mode of action,etc. of factors, agents, etc. that affect motor neuron developmentand/or contribute to a disease state, such as, without limitation, aneurodegenerative disease, e.g. ALS.

In certain embodiments, methods of the present invention employ humanmutant motor neurons that comprise a mutation in a gene associated witha neurodegenerative disease, for example ALS. One non-liming example ofa gene associated with the neurodegenerative disease ALS is SOD1. Avariety of SOD1 mutant alleles are known to be associated with ALS,including without limitation, SOD1G93A. In certain embodiments, methodsof the present invention utilize human motor neurons comprising a SOD1mutant allele (e.g., SOD1G93A) to screen for test agents that affectmotor neuron differentiation, development and/or survival. In certainembodiments, methods of the present invention comprise using such SOD1mutant motor neurons to identify a factor that has a non-cell autonomouseffect on the differentiation, development and/or survival of a motorneuron.

In certain embodiments, methods of the present invention employ humanmutant cells that are not motor neurons, which mutant cells comprise amutation in a gene associated with a neurodegenerative disease, forexample ALS. As but one non-limiting example, certain methods thepresent invention employ glial cells comprising a mutation in a geneassociated with a neurodegenerative disease such as ALS. In certainembodiments, methods the present invention employ glial cells comprisinga mutation in a SOD1 gene, such as, without limitation, SOD1G93A.

Those skilled in the art will be aware of other gene mutationsassociated with neurodegenerative diseases, and will be able to usemethods and compositions described herein to validate results of animalmodels, to identify novel factors, agents, etc. that affect motor neurondevelopment and/or contribute to a disease state, and/or to illuminatethe target, efficacy, toxicity, mode of action, etc. of factors, agents,etc. that affect motor neuron development and/or contribute to a diseasestate.

Non-Human Applications

Embryonic stem cells of the present invention and/or cells derived fromthem can be advantageously used in the study and/or modeling of humandiseases, although one of ordinary skill in the art will understand thatthe present disclosure is not limited to human applications. Thus, forexample, non-human embryonic stem cell lines may be used in the studyand/or modeling of diseases associated with pets (e.g., cats, dogs,rodents, etc.) as well as commercially important domestic animals (e.g.,cows, sheep, pigs, etc.). Additionally or alternatively, non-humanembryonic stem cell lines may be used to screen for agents that can beused in the prevention and/or treatment of diseases associated with petsand/or commercially important domestic animals.

EXAMPLES Example 1 Derivation of ES Cell Lines from the SOD1G93A ALSMouse Model

Embryonic stem cell lines were derived by crossing hemizygous micecarrying either the pathogenic (mutant) SOD1G93A transgene or thenon-pathogenic (wild-type) SOD1 transgene (Gurney et al., 1994) withhemizygous mice carrying a transgenic reporter gene in which greenfluorescent protein (GFP) expression is controlled by promoter elementsfrom the Hb9 gene (Hb9::GFP) (Wichterle et al., 2002). The Hb9 geneencodes a homeodomain transcription factor that is expressed inpostmitotic motor neurons (Arber, S. et al. Requirement for the homeoboxgene Hb9 in the consolidation of motor neuron identity. Neuron 23,659-74, 1999; Thaler, J. et al. Active suppression of interneuronprograms within developing motor neurons revealed by analysis ofhomeodomain factor HB9. Neuron 23, 675-87, 1999). This Hb9::GFPtransgene provides a marker for the differentiation of ES cells intomotor neurons (Wichterle et al., 2002). Blastocyst stage embryos wereretrieved from the progeny of these crosses and used to derive ES celllines that were genotyped using the polymerase chain reaction (PCR)(FIG. 1A,B). These analyses identified ES cell lines that carried onlythe Hb9::GFP transgene (Hb9GFP), lines that carried both Hb9::GFP andthe wild-type SOD1 transgene (SOD1), and a line that carried bothHb9::GFP and the mutant SOD1G93A transgene (SOD1G93A).

To determine whether these ES cells recapitulate the proper expressionpattern of the Hb9::GFP reporter transgene, we assessed GFP fluorescencein the undifferentiated ES cells and in chimeras created by injectingthese cells into non-transgenic blastocysts. Each of theundifferentiated cell lines lacked obvious GFP expression (For exampleFIG. 2A). However, in El 0.5 chimeras created with these cells, highlyspecific GFP expression was observed in the developing eye, hindbrainand spinal chord where Hb9 is known to be expressed (FIG. 1D) (Wichterleet al., 2002; Thaler et al., 1999). Immunostaining with antibodies thatpreferentially recognize the human SOD1 protein confirmed the PCRgenotyping of the cell lines and showed that both the SOD1 and SOD1G93Atransgenes are expressed in the undifferentiated ES cells (FIG. 1C).

Example 2 Production and Characterization of Motor Neurons by In VitroDifferentiation of SOD1G93A ES Cells

To determine whether pathogenic properties associated with ALS can berecapitulated in vitro, we generated motor neurons by differentiatingthe transgenic ES cell lines as previously described (Wichterle et al.,2002). Briefly, ES cells were dissociated into a single cell suspension,allowed to spontaneously aggregate into embryoid bodies (EBs) over 48hours and then treated with retinoic acid (RA) and soluble SonicHedgehog (Shh) protein¹ (Wichterle et al., 2002) for 5 days.

We found that the SOD1G93A genotype does not interfere with the initialspecification or differentiation of motor neurons, as no significantqualitative or quantitative differences were observed in thedifferentiation of the three cell lines. GFP expression in EBs derivedfrom the different cell lines, including SOD1 G93A, first appeared 5days after treatment with Shh and RA (FIG. 2A). Two days later, when EBswere dissociated with papain and plated, GFP positive cells with anobvious neuronal morphology could be observed (FIG. 2A). We usedfluorescence activated cell sorting (FACS) to determine the percentageof differentiating ES cells that expressed GFP and found nostatistically significant differences between the three cell lines(Hb9GFP 33%+/−4%, SOD1 33%+/−9%, SOD1G93A26%+/−6%) (see FIG. 6).

To confirm that the EB cells expressing GFP differentiated into bonefide motor neurons, we dissociated the EBs and performed immunostainingwith antibodies that recognize proteins known to be expressed in motorneurons (FIG. 2 B,C,D,E). As was previously observed with normal ES celllines (Wichterle et al., 2002), we found that GFP positive cells derivedfrom the SOD1G93A cell line expressed a neuronal form of tubulin (Tuj1,FIG. 2B), the transcription factors Hb9 and Isl1/2 (FIG. 2 C,D; FIG. 7)and the enzymatic machinery required to generate acetylcholine (Chat,FIG. 2E).

Example 3 The SOD1G93a Genotype Affects the Survival of Motor Neurons inCulture

ALS is a late onset, progressive neurodegenerative disease, and micecarrying the human SOD1G93A transgene develop symptoms as a consequenceof motor neuron loss after several weeks. Therefore, it seemed possiblethat motor neurons derived from ES cells might display neurodegenerativeproperties only after they have been maintained in culture for aprolonged length of time. To determine the period of time that ES cellderived motor neurons can survive in culture, we dissociated day 7Hb9GFP and SOD1G93A EBs and plated the resulting mixture of GFP positiveand negative cells at two different densities in the presence ofneurotrophic factors (Wichterle et al., 2002) (FIG. 3). We observed thatthe number of GFP positive cells decreased precipitously during thefirst two weeks, and then continued to decrease over the followingweeks. However, GFP positive cells could still be detected in bothHB9GFP and SOD1G93A derived cultures 54 days after plating (FIG. 3A).Although GFP positive cells were present in both cultures at later timepoints, the number of cells expressing GFP decreased more rapidly in theSOD1G93A (FIG. 3C) cultures than in Hb9GFP control cultures (FIG. 3B).

To confirm the affect of the SOD1G93A genotype on the number of GFPpositive cells, we differentiated both the SOD1G93A and Hb9GFP ES cellsinto motor neurons, plated equal numbers of cells at two differentconcentrations (8×10⁵ (n=3) and 4×10⁵ (n=3) EB cells per well) andcounted the number of GFP positive neurons in the cultures at 2 and 4weeks (FIG. 3D,E,F,G). Under both plating conditions, significantlyfewer GFP positive cells were observed in the SOD1G93A cultures at both2 and 4 weeks (FIG. 3D,E,F,G).

Example 4 Histopathological Hallmarks of ALS can be Observed in ESCell-Derived Motor Neurons

Hb9GFP and SOD1G93A ES cells differentiate into motor neurons at asimilar efficiency, but the cultures show differences in the number ofGFP positive cells over time. Thus, a pathological process may underliethe preferential loss of GFP positive cells in the SOD1G93A cultures. Toinvestigate these processes and to determine whether they mirror eventsthat occur during the progression of ALS, we examined motor neurons inculture for the presence of histopathological hallmarks of the disease.Motor neurons in ALS patients and transgenic mice carrying the SOD1G93Aallele accumulate protein inclusions that are recognized by antibodiesspecific to the SOD1 protein (Boillee et al., 2006; Bruijn et al.,19970. We therefore determined whether aggregation of the mutant SOD1protein accompanies the loss of GFP positive motor neurons in SOD1G93Acultures by staining with antibodies specific to the human SOD1 proteinat 7, 14 and 21 days following EB dissociation (FIG. 4A). At 7 and 14days following dissociation both the wild-type SOD1 protein and themutant SOD1G93A protein were localized broadly and evenly in thecytoplasm of GFP positive motor neurons (FIG. 4B,A). However, at 14 dayspunctate structures staining brightly with the SOD1 antibody could beobserved in a small proportion of motor neurons expressing the SOD1 G93Aprotein, (FIG. 4A).

When cultures were examined 21 days after dissociation, a shift inprotein localization was observed in the SOD1G93A motor neurons (FIG.4A,D). In 43/54 (79.75±7.75%) of the motor neurons selected at randomfor analysis by GFP expression, the SOD1 G93A protein localized toinclusions in the perinuclear space, in the cell body and also in theneural processes (FIG. 4A,D supplemental FIG. 3). When control motorneurons expressing wild-type SOD1 were examined 21 days afterdifferentiation (FIG. 4B,D), we observed inclusions in a smallerproportion of cells 23/64 (35.93±0.43%). The inclusions in motor neuronsexpressing the SOD1G93A allele were also significantly larger in area(FIG. 8A), significantly longer (FIG. 8B) and displayed a higher opticaldensity, suggesting that they contained more SOD1 protein at a higherconcentration (FIG. 8C).

The levels of ubquitinated proteins are significantly elevated in motorneurons of ALS patients and SOD1G93A transgenic animals during neuraldegeneration (Bruijn et al., 1997; Ince, P. G., et al., Amyotrophiclateral sclerosis associated with genetic abnormalities in the geneencoding Cu/Zn superoxide dismutase: molecular pathology of five newcases, and comparison with previous reports and 73 sporadic cases ofALS. J Neuropathol Exp Neurol 57, 895-904, 1998; Wang, J. et al.Copper-binding-site-null SOD1 causes ALS in transgenic mice: aggregatesof non-native SOD1 delineate a common feature. Hum Mol Genet 12,2753-64, 2003; Watanabe, M. et al. Histological evidence of proteinaggregation in mutant SOD1 transgenic mice and in amyotrophic lateralsclerosis neural tissues. Neurobiol Dis 8, 933-41, 2001). Examination ofthe ES cell-derived motor neurons revealed an increase in staining withanti-ubiquitin antibodies, relative to other cells in the culture. Thisstaining often colocalized with the SOD1 protein inclusions (FIG. 4C).

The death of motor neurons in ALS patients and transgenic mice carryingmutant SOD1 genes occurs through activation of programmed cell deathpathways. Apoptosis in these cells has been proposed to be mediatedthrough the release of cytochrome c and the activation of caspase-3(Pasinelli, P., Houseweart, M. K., Brown, R. H., Jr. & Cleveland, D. W.Caspase-1 and -3 are sequentially activated in motor neuron death inCu,Zn superoxide dismutase-mediated familial amyotrophic lateralsclerosis. Proc Natl Acad Sci USA 97, 13901-6, 2000; Raoul, C. et al.Motoneuron death triggered by a specific pathway downstream of Fas.potentiation by ALS-linked SOD1 mutations. Neuron 35, 1067-83, 2002).Examination of the SOD1G93A motor neurons 14 days after dissociation ofEBs, revealed that some neurons expressed activated caspase-3 andcontained diffuse cytoplasmic staining with cytochrome c specificantibodies (FIG. 9A,B,C). Thus, the SOD1G93A motor neurons appear toinitiate cell death pathways in vitro that are similar to thoseactivated in vivo during the course of disease (Pasinelli et al., 2000).

Example 5 SOD1G93A Glial Cells Adversely Affect Survival of ES CellDerived Motor Neurons

Both autonomous defects in motor neurons and toxic non-cell autonomousinteractions with other cell types in the spinal cord have beenimplicated in ALS pathology (Bruijn et al., 2004; Clement et al., 2003;Boillee et al., 2006). Only a subset of cells in EBs became motorneurons under our differentiation conditions. We therefore consideredthe possibility that cells within the EBs might also develop into othercell types that normally associate with neurons in the spinal cord.These additional cells might, as suggested by chimeric experiments,contribute non-cell autonomously to the loss of neurons in SOD1G93Acultures. Glial cells are closely associated with motor neurons, andboth are derived from a common progenitor in vivo (Zhou, Q. & Anderson,D. J. The bHLH transcription factors OLIG2 and OLIG1 couple neuronal andglial subtype specification. Cell 109, 61-73, 2002). We thereforeaddressed the possibility that glial cells are present in our cultures.To determine if these cells, were also produced by our differentiationprotocol, we stained SOD1G93A cultures with antibodies specific to theglial fibrillary acidic protein (GFAP) (Bignami, A. & Dahl, D.Astrocyte-specific protein and neuroglial differentiation. Animmunofluorescence study with antibodies to the glial fibrillary acidicprotein. J Comp Neurol 153, 27-38, 1974). Indeed, GFAP positive cellswere found in close association with GFP positive motor neurons (FIG.2F). We found that approximately 30% of the cells at 28 days were GFAPpositive (Hb9GFP: 31%, SOD1: 32%, SOD1G93A: 36%). Thus, it seemedpossible that the SOD1G93A glial cells in these cultures might beadversely affecting motor neuron survival.

To examine this possibility, we differentiated the three ES cell lines(Hb9GFP, SOD1 and SOD1 G93A) into motor neurons and plated them onestablished monolayers of primary glia isolated from the cortex ofneonatal mice with differing SOD1 genotypes (wild-type SOD1, and themutant SOD1G93A) (Banker, G. & Goslin, K. Culturing nerve cells, xii,666, 11 of plates (MIT Press, Cambridge, Mass., 1998). During the firstseven days after plating on either glial monolayer, motor neurons of allgenotypes increased in size and took on a more mature morphology (FIG.5A). However, by 14 days after plating there was a 50% decrease in thenumber of wild-type SOD1 derived motor neurons in co-cultures withSOD1G93A glia compared to the same preparation of neurons plated onwild-type SOD1 glia. Similarly, we did not see a significant reductionin the number of Hb9GFP motor neurons when plated on SOD1 glia, but didsee a reduction of 30% if the same neurons were co-cultured withSOD1G93A glia (FIG. 5B). These data suggest that wild-type SOD1 gliaprovide a permissive environment for motor neuron growth anddifferentiation, comparable to other well-established in vitro systemsfor motor neuron culture (Ullian, E. M., Harris, B. T., Wu, A., Chan, J.R. & Barres, B. A. Schwann cells and astrocytes induce synapse formationby spinal motor neurons in culture. Mol Cell Neurosci 25, 241-51, 2004;Allen, N. J. & Barres, B. A. Signaling between glia and neurons: focuson synaptic plasticity. Curr Opin Neurobiol 15, 542-8, 2005; Ullian, E.M., Christopherson, K. S. & Barres, B. A. Role for glia insynaptogenesis. Glia 47, 209-16, 2004). In contrast, when we co-culturedwild type motor neurons (SOD1 or Hb9GFP) with glia from SOD1G93A mice,we observed a marked reduction in survival (FIG. 5 A,B).

We next investigated the affect of the presence of the mutant SOD1transgene in motor neurons in the co-culture system. When mutantSOD1G93A motor neurons were plated on wild-type SOD1 glial cells, therewas a 27% decrease in the number of neurons between 7 and 14 days. Theloss of motor neurons increased to 75% when the mutant SOD1G93A motorneurons were cultured with mutant SOD1G93A glia (FIG. 5 A,B).

Together, our results show that the SOD1G93A genotype in glial cells hasa negative effect on motor neuron survival regardless of the motorneuron genotype. However, a greater negative effect is observed with theSOD1G93A motor neurons. Thus, glial cells have a non-cell autonomouseffect on motor neuron survival and mutant motor neurons are moresensitive to the effect.

To determine whether the differing influences of the wild-type SOD1 andthe mutant SOD1 G93A glial cell cultures could be explained by thepresence of different proportions of distinct glial cell types, wecharacterized the two populations by immunostaining with establishedglial markers. We did not observe a significant difference in thewild-type SOD1 and the mutant SOD1G93A glial populations over time (FIG.10).

Example 6 Methods

Examples 1 through 5 were performed using the following methods:

Derivation of Mouse Embryonic Stem Cells.

ES cell lines were derived from crosses between mice transgenic forHb9:GFP (Jackson lab, Stock Number:005029) and mice transgenic forSOD1^(G93A) (Jackson lab, Stock Number:004435) or SOD1^(WT) (Jacksonlab, Stock Number:002297). Transgenic Hb9:GFP females were injected IPwith 7.5 units of pregnant mares' serum (Calbiochem) followed 46-50 hlater with 7.5 units of human chorionic gonadotropin (HCG) (Calbiochem).After administration of HCG, females were mated with either SOD1 G93A orSOD1 transgenic males. Females were scarified three days later andblastocysts were flushed from the uterine horn with mES cell media(Knockout-DMEM (GIBCO), 15% Hyclone Fetal Bovine Serum (Hyclone), 10.000unit Penicillin and 1 mg/ml Streptomicin (GIBCO), 2 mM Glutamine(GIBCO), 100 mM non-essential aminoacid (GIBCO), 55 mMbeta-mercapto-ethanol (GIBCO), 1,000 units/ml leukocyte inhibitingfactor (Chemicon)). Blastocysts were then plated individually into one10 mm-well of a tissue culture dish containing a feeder layer ofmitotically inactivated mouse embryonic fibroblast, in the presence ofmES cell media supplemented with the MEK kinase inhibitor PD98059 (cellsignaling, inc). 48 hours after plating embryos, one half volume offresh media was added to each culture well. Starting three days afterplating, the culture media was changed.

Four to five days after plating, ICM-derived outgrowth were observed,dislodged from the rest of the cells with a pasteur pipette, washed oncein a drop of PBS and then incubated for 10 minutes in a drop of 0.25%trypsin at 37 C. The ES cells clumps were then gently dissociated with aPasteur pipette filled with mouse ES cell media and transferred onto afresh layer of fibroblasts in a 10 mm tissue culture well. For routineculture, the mouse ES cells are generally split 1:6 with a solution of0.25% trypsin (GIBCO) every 2-3 days.

Generation of Chimeric Embryos.

Chimeric embryos were generated as previously described (Hogan, B.Manipulating the mouse embryo: a laboratory manual, xvii, 497 p. (ColdSpring Harbor Laboratory Press, Plainview, N.Y., 1994). Blastocysts werecollected from the uterus of non-transgenic pregnant females mice 3.5days postcoitum. The ES cells were injected (about 10 for eachblastocyst) using a microinjection pipette with a diameter of 12-15 μmapplying a brief pulse of the Piezo (Primetech, Ibaraki, Japan) on oneside of the blastocyst and pushing the needle through the zona andtrophectoderm layer into the blastocoel cavity. Ten injected blastocystswere transferred to each uterine horn of 2.5 days postcoitumpseudopregnant Swiss females that had mated with vasectomized males.Recipient mothers were sacrificed at 10.5 days postcoitum, and embryoswere quickly removed from the uterus and placed in a dish with cold PBSfor whole mount analysis of GFP fluorescence.

Differentiation of mES Cells into Motor Neurons.

Mouse ES cells were differentiated into motor neurons according tomethods previously described (Boillee et al., 2006). The ES cells weregrown at 70-80% confluence in a 10 cm plate (Falcon) in mouse ES cellsmedia. To form EB's, cells were washed once with a PBS solution toeliminate the traces of media and then incubated with 1 ml of 0.25%trypsin (GIBCO) for 5-10 minutes at room temperature. After this thecells were resuspended in 10 ml of DM1 media (DMEM-F12 (GIBCO), 10%Knockout serum (GIBCO), pen strep, glutamine (GIBCO) and2-mercaptoethanol (GIBCO)), they were counted and plated at aconcentration of 200.000/ml in Petri dishes (Falcon). After 2 days theembryoid bodies were split from 1 dish into 4 new Petri dishescontaining DM1 medium supplemented with RA (100 nM μM; stock: 1 mM inDMSO, Sigma) and sonic hedgehog (300 nM, R&D Systems). The media ischanged after 3-4 days. On day 7 the embryoid bodies were dissociated insingle cells suspension. The EBs were collected in a 15 ml falcon tube,centrifuged at 1000 rpm for 5 min, washed once with PBS and incubated inEarle's Balanced Salt Solution with 20 units of papain and 1000 units ofDeoxyribonuclease I (Worthington Biochemical Corporation) for 30-60minutes at 37° C. The mixture was then triturated with a 10 ml pipetteand centrifuged for 5 minutes at 1000 RPM. The resulting cell pellet waswashed once with PBS and finally resuspended in supplemented F12 media(F12 medium (GIBCO) with 5% horse serum (GIBCO), B-27 supplement(GIBCO), N2 supplement (GIBCO)) with neurotrophic factors (GDNF, CNTF,and BDNF (10 ng/ml, R&D Systems)). The cells were then counted andplated on Poly-D-Lysine/Laminin CultureSlides (BD biosciences) or on alayer of primary glia cells. For the motor neurons survival experiments,GFP positive cells with visible axons and dendrites were counted atdifferent time points after plating (7, 14, 21, 28 days).

Polymerase Chain Reaction.

All PCR reactions were performed using an MJ Research Thermal Cycler,and TaKaRa Ex Taq HS (Takara) enzymes. For the SOD1 genotyping of thenewly derived ES cell lines and transgenic mice the forward primer: CATCAG CCC TAA TCC ATC TGA (SEQ ID NO:1) and reverse primer: CGC GAC TAACAA TCA AAG TGA (SEQ ID NO:2) amplified a 236 bp fragment in the 4^(th)exons of the gene. As an internal control a set of primers thatamplified a 324 bp fragment of the IL-2 gene (forward: CTA GGC CAC AGAATT GAA AGA TCT (SEQ ID NO:3), reverse: CAT CAG CCC TAA TCC ATC TGA (SEQID NO:4)) were used. The annealing temperature for this reaction was 60°C. for 35 cycles. For GFP a set of primers (forward: AAG TTC ATC TGC AACACC (SEQ ID NO:5), reverse: TCC TTG AAG AAG ATG GTG CG (SEQ ID NO:6))that amplified a fragment of 173 bp of the gene were used, with anannealing temperature of 60° C. for 35 cycles.

FACS.

For FACS analysis a BD biosciences LSRII flow cytometer was used. Theembryoid bodies were dissociated with papain and resuspended in cold PBSwith 2% FBS, Calcein blue (Invitrogen) was used to assay the cellviability. The cells were then analyzed using a non transgenic mouseembryonic stem cell lines as a negative control. The FACS Diva softwarepackage (BD Biosciences) was used for data analysis

Glia Cultures.

Glia monolayers were obtained from P2 mice born from matings betweentransgenic SODG93A. Tissue was isolated in Calcium and MagnesiumFree-Hanks's BSS (HBSS). Under a dissecting microscope the cortex wasisolated and carefully striped of the meninges. The tissue was split insmall pieces then transferred to a 50 ml centrifuge tube in a finalvolume of 12 ml of HBSS. Tissue digestion was performed usingtrypsin—EDTA (GIBCO BRLno.25200) and 1% DNAse (Sigma no. DN-25) at 37°C. for 15 min, swirling the mixture and periodically. We collected thedissociated tissue and triturated using a fire polish Pasteur glasspipette and filtered the combined supernatant through a 72 gm nylon mesh(NITEX 100% polyamide Nylon Fiber TETKO Inc.) to remove anyundissociated tissue. The filtered material was centrifuged at 1000 rpmfor 5 min to pellet the cells, resuspend in 2 ml of Glia medium (MinimumEssential Medium with Earl's salts, GIBCO BRL no. 11095-080, 20%Glucose, Penicillin-streptomycin, GIBCO BRL no. 15145-014, and 10% HorseSerum GIBCO BRL no. 26050-070) and cell number was counted. The yieldfrom one brain was generally enough to plate one T75 flask (Falcon no.3084). Once monolayers were confluent (generally in 10 to 14 days) cellswere replated on 24 or 12 well multiwell dishes over poly-D-Lysine (0.5mg/ml for 30 min RT) coated glass cover slips.

Immunocytochemistry Analysis.

The cells were fixed with 4% paraformaldehyde-PBS, blocked andpermeabilized with BSA (1%)-Triton X100 (0.1%). After incubatingovernight with the following antibodies: mouse monoclonal anti-Tuj1(Covance), Islet 1 Islet2, RC2 (Developmental Studies Hybridoma BankUniversity of Iowa, IA, USA) anti-sod1 (SIGMA), S100 (Chemicon), CNPase(Abeam) and rabbit anti-HB9 (Tom Jessell, Columbia University), GFAP(Chemicon) anti-ubiquitin (DAKO); goat anti-Vimentin (Chemicon); ratanti-CD 11 b (Abeam) (see Table 1)

Table 1 describes primary antibodies used. Antiserum (host species),working dilution and source.

Antiserum Dilution Source Tuj 1(anti-Mouse) 1/1000 Covance Islet1(anti-Mouse) 1/1 Hybridoma Bank ChAT (anti-Goat) 1/100 Chemicon Hb9(anti-Rabbit) 1/1000 Jessel Lab GFAP (anti-Rabbit) 1/1000 Chemicon S100(anti-Mouse) 1/100 Chemicon RC2 (anti-Mouse) 1/1 Hybridoma Bank Vimentin(anti-goat) 1/100 Chemicon CD 11b (anti-Rat) 1/100 Abcam hSOD1(anti-Mouse) 1/200 Sigma Caspase 3(anti-Rabbit) 1/2000 BD PharmingenUbiquitin (anti-Rabbit) 1/200 DAKO Cytochrome C (anti-Mouse) 1/200 Abcam

The cells were then incubated with Donkey anti-rabbit conjugated to Cy3(1:100; 2 h) and Donkey anti mouse conjugated to Cy5 second antibodies(1:100; 2 h; Jackson ImmunoResearch (West Grove, Pa., USA.). Aftermounting the samples in Vectashield (Vector Labs, Burlingame, Calif.,USA), confocal or epi-fluorescent microscopy was performed using OlympusFV 1000, 40× and 60× oil immersion objective 1.45 NA or fluorescentmicroscope Olympus IX70. Image acquisition was performed using FLUOVIEWsoftware 4.0 for relative fluorescence analysis, all settings such asexposure time, magnification and gain were maintained constant for allsamples. Offline analysis of relative intensities for all the sampleswas done using Metamorph 4.5 (Downingtown, Pa., USA). Only cells havingmorphological features of neurons (i.e., phase bright soma and severalneurites) were considered for subsequent analysis.

Neuronal Density.

HB9-GFP positive motor neurons were counted for condition studied (Zeissmicroscope, 40× 1.3 NA, oil immersion objective). The density ofneurons, normalize as a percent of initial number counted at 7 DIV or 14DIV was established for all conditions studied. These experiments werecarried out three times as independent experiments.

Data Analysis.

The data was obtained from control and SOD1 G93A motor neurons inparallel conditions (sister plates) to reduce dispersion. Statisticalanalysis were performed using Student's t-Test or ANOVA and areexpressed as arithmetic mean±S.E.M.; t-test values of * P<0.05, **P<0.01, *** P<0.005 were considered statistically significant. Each setof data presented was performed in sister cultures to reducevariability. Similar significances were found expressing the data incumulative distributions plots. Therefore, we chose to present the dataas mean±E.M. to simplify its presentation. The kinetic analysis was doneusing MiniAnalysis 5.0 (Synaptosoft).

Example 7 Human ES cell lines are sensitive to SOD1G93A glia

To generate a large supply of motor neurons from human ES cells for thestudy of ALS we adapted a recently reported method for the production ofthese cells within embryoid bodies (EBs) (Singh Roy, N. et al.Enhancer-specified GFP-based FACS purification of human spinal motorneurons from embryonic stem cells. Exp Neurol. December; 196(2):224-34,2005) (FIG. 11 a). Undifferentiated, self-renewing HuES 3 ES cells(Cowan, C. A. et al. Derivation of embryonic stem-cell lines from humanblastocysts. N Engl J Med March 25; (13):1353-6, 2004) were dissociatedinto small clumps using collagenase treatment and then allowed tospontaneously differentiate in suspension for 14 days (FIG. 11 a, b).Staining of the resulting EBs with the neuronal progenitor marker PAX6(FIG. 11 b) demonstrated that a substantial percentage (29%+/−16%, FIG.15 a, b) contained cells differentiating down the neuronal lineage. Todirect these progenitors towards an anterior and ventral motor neuronidentity, we cultured the EBs another 14 days in the presence ofretinoic acid (RA) and a small molecule agonist of the sonic hedgehog(SHH) pathway. Under the influence of these morphogens the population ofPAX6 positive progenitors expanded (45%+/−15%; FIG. 1 b; FIG. 15 a, b)and expression of the ventral progenitor markers NKX6.1 and ISL1/2 wasinduced (FIG. 11 b; FIG. 15 a,b). To promote motor neurondifferentiation and survival, we then transferred these 28 day-old EBsto media containing neurotrophic factors for a final 14 days. After 42days of differentiation, the number of progenitors expressing PAX6 andNKX6.1 had begun to decline (FIG. 15 a, b), while the number of cellsexpressing ISl1/2 continued to increase (FIG. 11 b; FIG. 15 a, b). Inaddition, expression of the HB9 transcription factor, which is expressedin maturing post-mitotic motor neurons, was detected in 8% of all cells(FIG. 11 b; FIG. 15 a, b). Furthermore, when plated on laminin, theseEBs elaborated impressive neuronal processes (FIG. 11).

To further characterize the putative motor neurons contained withinthese EBs, the 42 day-old EBs were dissociated with papain and theresulting cells plated directly onto glial mono layers prepared from thecortex of neonatal mice (FIG. 16). We found, as had been previouslyreported with neurons derived from mouse ES cells (Song, H. et al.Astroglia induce neurogenesis from adult neural stem cells. Nature. May2; 417(6884):39-44, 2002) that culturing human ES cell derived neuronson a glial monolayer promoted their survival (FIG. 16 b, c). Co-stainingof cells with antibodies specific to a neuronal form of tubulin (Tuj1)and the transcription factors Hb9 and Isl1/2 (FIG. 17 a, b), as well asco-staining for Hb9 and choline acetyl transferase (Chat) (FIG. 17 c)confirmed that many neurons isolated from these EBs were differentiatingtowards a motor neuron identity.

To ensure that the appearance of motor neurons within these EBs wasdependent on the influences of RA and SHH, we repeated ourdifferentiation scheme in the absence of one or both of these morphogensand counted the number of HB9 positive cells (FIG. 11 c). When SHH or RAactivity were removed individually, the frequency of cells expressingHB9 fell to 0.7% (+/−0.2) and 1.1% (+/−0.5%) respectively. If bothsignaling molecules were omitted, less than 0.2% of the dissociatedcells expressed HB9 (0.17%+/−0.07%). We further confirmed the robustnessof our approach for generating motor neurons by differentiating sixindependent human ES cells lines and then quantifying the number of HB9positive cells within the resulting EBs (FIG. 11 d). We found thatHuES1, HuES3, HuES5 and HuES9 ES cell lines all differentiated with asimilar efficiency (HuES 1: 7.1%+/−1.8%; HuES 3: 8.5%+/−0.5%; HuES 5:4.7%+/−0.8%; HuES 9: 7.7%+/−1.5%), while HuES 12 cells differentiated ata lower efficiency (2.8%+/−1.3%) and HuES 13 cells at a higherefficiency 13.9% (+/−3.8%). These results are consistent with a recentreport that suggests independent human ES cell lines can have varyingabilities to differentiate into certain cell types (Osafune, K. et al.Marked differences in differentiation propensity among human embryonicstem cell lines. Nat Biotechnol. March; 26(3):313-5, 2008).

In order to identify living motor neurons in cultures of differentiatinghuman ES cells, we generated a stable transgenic human ES cell line inwhich sequences coding for the green fluorescent protein (GFP) wereunder the control of the murine Hb9 promoter (Wichterle et al., 2002)(FIG. 12). To validate that this transgenic cell line accuratelyreported HB9 expression, we differentiated the cells, plated them onglial monolayers and co-stained with antibodies specific to GFP and HB9.HB9 expression was observed in 95% of GFP positive cells (FIG. 12 c;FIG. 18 a-d). We next investigated whether these GFP positive cellsexpressed other markers that would be consistent with a maturing motorneuron identity (FIG. 12; FIG. 18). We observed considerable overlapbetween GFP and expression of NKX6.1 (FIG. 180 but no co-expression withNKX2.2 (FIG. 18 e), confirming that GFP positive cells had acquired thecorrect dorsal-ventral identity (Jessell TM. Neuronal specification inthe spinal cord: inductive signals and transcriptional codes. Nat RevGenet. October; 1(1):20-9, 2000). Additionally these cells expressedISL1/2 (FIG. 12 e) and ChAT (FIG. 12 f) but no longer expressed theprogenitor marker PAX6 (FIG. 12 d). Antibody co-staining experimentsalso demonstrated that GFP positive cells did not co-express makersfound in other neuronal subtypes such as the interneuron markers CHX10(FIG. 18 h) and LHX2 (FIG. 18 g).

The results that we have described thus far confirm that it is possibleto reproducibly generate a large supply of human motor neurons fromembryonic stem cells. We next sought to use these human neurons to askwhether they, like their mouse counterparts, are sensitive to thenon-cell autonomous effect of glial cells overexpressing a mutant SOD1gene product. To this end, we dissociated 42 day-old EBs and plated theresulting cells on primary glial monolayers derived from either SOD1G93Atransgenic or control mice (FIG. 13 a). After 10 days a significantdifference (p<0.05) in the number of HB9 positive motor neurons was seenbetween the two culture conditions (FIG. 13 b) is already appreciable.In cultures containing SOD1 G93A glia less than half as many motorneurons remained (131+/−53) as in cultures containing non-transgeniccontrol glia (269+/−44) (FIG. 13 b). The deficit in motor neuronsurvival in co-cultures with SOD1G93A glia became even more pronouncedafter 20 days (FIG. 13 c, d). We next sought to confirm that the toxiceffect of glia we observed in our initial experiments was due to theaction of the mutant SOD1 protein rather than mere SOD1 proteinover-expression. Motor neuron preparations were generated from theHb9::GFP human ES cell line and co-cultured for 20 days withnon-transgenic glia or glia which either over-expressed the wild-typehuman SOD1 protein or the mutant SOD1G93A protein (FIG. 13 e-h). Therewas no discernable difference between the number of GFP positive motorneurons present in culture with the non-transgenic Glia (304+/−60; FIG.13 e, h) or with glia over-expressing the wild type SOD1 protein(328+/−30; FIG. 13 e, g). In contrast, there was a highly significantreduction (p<0.01) in the number GFP positive motor neurons (127+/−16;FIG. 13 e, f) present in culture with the SOD1G93A Glia, confirming thatthe non-cell autonomous effect of glia was mediated through the mutantSOD1 protein.

In both patients and mice carrying mutant alleles of the SOD1 gene,intracellular aggregation of the SOD 1 protein is often documented andhas been associated with motor neuron death (Boillee et al., 2006). Wetherefore wondered whether the toxic effect of glial cells expressingthe mutant SOD1 protein that we observed was a downstream consequence ofprotein aggregation. To address this, we separately cultured primarymouse glia and mouse ES cell derived motor neurons carrying the sameSOD1G93A transgene and stained the cultures with antibodies specific forthe human SOD1 protein. After 21 days in culture, the SOD1 protein inmouse motor neurons was observed to aggregate into cytoplasmic andperinuclear inclusions (FIG. 19 a) (Di Giorgio, F. P., Carrasco, M. A.,Siao, M. C., Maniatis, T. & Eggan, K. Non-cell autonomous effect of gliaon motor neurons in an embryonic stem cell-based ALS model. Nat.Neurosci. 10, 608-614, 2007). In contrast, even after more than 90 daysin culture, the SOD1 protein was found to be broadly and diffuselylocalized in the cytoplasm of all glial cells (FIG. 19 b), suggestingthat the mutant protein is mediating its effect in these cells through amechanism independent of protein aggregation.

ALS leads to the specific degeneration of motor neurons. Therefore, ifthe toxic effect of glial cells that we have observed is relevant to ALSthen we might expect that other spinal cord neuronal types such asinterneurons would not be sensitive to it. During our characterizationof human ES cell derived motor neurons we noted that additional neuronsexpressing the transcription factors CHX10 and LHX2, indicative of V2and D1 interneuron differentiation, were also produced (FIG. 18 g, h).To test whether these neuronal types were affected by co-culture withmutant glia, we dissociated 42 day-old EBs, plated equal numbers ofcells on either SOD1G93A glia or non-transgenic glia (FIG. 14 a-e) andafter 20 days of culture stained for Tuj1 and either LHX2 (FIG. 4 d) orCHX10 (FIG. 14 e). We found that neurons expressing either of theseinterneuron markers were unaffected by culture with mutant glia (FIG. 14b, c), in striking contrast to the sensitivity of motor neurons to thisculture environment.

To determine if the toxic effect of mutant glial cells was theconsequence of a specific activity within this cell type rather then ageneral property of any cell over expressing the SOD1G93A mutation, weplated motor neuron preparations on mouse embryonic fibroblasts (MEFs)prepared from SOD1G93A and non-transgenic sibling embryos (FIG. 14 f-h).After 20 days of co-culture we did not observe a significant differencebetween the number of HhB9, Ttuj1 double positive motor neurons presenton SOD1G93A MEFs (204+/−28) or non-transgenic MEFs (197+/−23) (FIG. 14g, h), consistent with the hypothesis that astrocytes are specificallyresponsible for the non-cell autonomous effect we observed (Di Giorgioet al., 2007; Nagai et al., 2007).

Example 8 Methods

Example 7 was performed using the following methods:

Growth of Human Embryonic Stem Cells.

The HuES cell lines were obtained from Doug Melton and cultured asdescribed by Cowan et al. (Cowan et al., 2004). The hESCs weremaintained on a feeder layer of inactivated mouse embryonic fibroblasts(GlobalStem) in human ES cell media (KO-DMEM (Gibco), 10% KO SerumReplacement, 10,000 units Penicillin and 1 mg/ml Streptomicin (GIBCO), 2mM Glutamine (GIBCO), 100 μM non-essential amino acids (GIBCO), 55 μMbeta-mercapto-ethanol (GIBCO), 10% Plasmanate (Bayer), 10 ng/mL bFGF2(GIBCO)). The cells were cultured at 37° C. and 5% CO₂. Media wasreplaced daily for the duration of hESC expansion and the cells in theseconditions were passaged every 5-7 days using a solution with 0.05trypsin (GIBCO).

Differentiation of Human Embryonic Stem Cells into Motor Neurons.

For differentiation into motor neurons, the cells were allowed to reach80-90% confluency, washed once with PBS, and then incubated for 15minutes at 37° C. in a solution of 1 g/L Collagenase IV (GIBCO) inDMEM-F12 (GIBCO).

Using a cell scraper, the ES cell colonies were scraped and washed offthe plate, centrifuged for 5 minutes at 1000 RPM and resuspended inhuman ES cell media without bFGF2 or plasmanate in low attachment 6-wellplates.

After 24 hours, the cells had aggregated to form embryoid bodies (EBs),and the media was changed to remove debris by centrifuging the EBs andresuspending in fresh in human ES cell media without bFGF2 or plasmanatein low attachment 6-well plates. EBs were cultured as such for 13 moredays, with half of the media changed every two days, and a completemedia change every week. After 14 days, the EBs were induced toward acaudal and ventral identity using retinoic acid (1 μM, Sigma) and anagonist of the Shh signaling pathway (1 μM) in N2 media: 1:1DMEM:F-12+Glutamate (Gibco), 10,000 units Penicillin and 1 mg/mLStreptomicin (Gibco), 1% N2 Supplement (Gibco), 0.2 mM ascorbic acid(Sigma-Aldrich), 0.16% D-(+)-Glucose (Sigma-Aldrich), BDNF (10 ng/ml,R&D Systems), for another 14 days. The EBs were then matured for a final14 days in N2 media with GNDF (10 ng/mL, R&D Systems). After 42 days ofdifferentiation, the EBs were dissociated. To dissociate the EBs, theywere centrifuged at 1000 rpm for 5 min in a 15 ml falcon tube, and thenwashed once with PBS to eliminate residual media. The EBs were thenincubated for 60 minutes at 37° C. in Earle's Balanced Salt Solutionwith 20 units of papain and 1000 units of Deoxyribonuclease I(Worthington Biochemical Corporation). EBs were triturated using a 2 mLserological pipette every 15-20 minutes during this incubation. When analmost single cell suspension was achieved, the cells were centrifugedfor 5 minutes at 1000 RPM. The resulting cell pellet was washed oncewith PBS and then resuspended in N2 media with neurotrophic factors(GDNF, and BDNF (10 ng/ml, R&D Systems)). These cells were then countedand plated on Poly-D-Lysine/Laminin CultureSlides (BD biosciences) or ona layer of primary glial cells. Depending on the experiment, motorneurons or intemeurons were counted 10 or 20 days after plating.

Generation of the HuES 3 Hb9::GFP Cell Line.

To generate the Hb9::GFP HuES 3 cell line, HuES 3 cells wereelectroporated with a plasmid containing a neomycin resistance cassetteand the coding sequence of Green Fluorescent Protein undertranscriptional control of a 9 kb murine Hb9 promoter restrictionfragment. The plasmid was a kind gift of Hynek Witcherle (ColumbiaUniversity) and was a modification of the construct described inWitcherle et al. (2002). The elctroporation was performed as describedin Zwaka P T. et al. (Zwaka T P, Thomson J A. Homologous recombinationin human embryonic stem cells. Nat Biotechnol. March; 21(3):319-21,2003).

Undifferentiated HUES 3 cells were grown as described below. Once thecells reached 80-90% confluency, they were dissociated in trypsin andcounted. Approximately 1.0×10⁷ were resuspended in 0.7 mL of human EScell media and mixed with 0.1 mL of the same media containing 30 μg oflinearized vector. This mix of cells and DNA was then transferred to a0.4 cm cuvette and exposed to a pulse of 320 V, 200 μF at roomtemperature. After 10 minutes at room temperature the cells were platedon a 10 cm dish of MEF, and 48 hours after electroporation the cellswere switched to media containing G418 (50 μg/mL, GIBCO). Selectionmedia was changed daily for 14 days, after which we picked and expanded24 resistant human ES cell colonies. In order to assay GFP expression,we differentiated six of these resistant clones into motor neurons andimmunostained for GFP and HB9 co-expression. Two of these clones gaverise GFP positive cells that elongate green axons, however only oneclone was validated by immunoreactivity to the Hb9 antibody and used insubsequent experiments.

Immunocytochemistry Analysis.

Cells were fixed with 4% para-formaldehyde for 30 minutes at roomtemperature. After fixation, the cells were washed 3 times with PBS for10 minutes and then treated for 1 hour in a blocking solution (PBS(Cellgro), donkey serum (10%, Jackson Immunoresearch)) plus Triton X (0A%, Sigma) for permeabilization. After blocking, the cells were incubatedovernight at 4° C. with primary antibodies: mouse anti-beta tubulin III(Covance); rabbit anti-beta tubulin III (SIGMA); Pax6, Nkx6.1, Nkx2.2,Isl 1, Hb9 (DSHB); Chx10, Lhx2 (Santa Cruz Technologies); ChAT(Chemicon); rabbit anti-GFP conjugated Alexa fluor 488 (MolecularProbes); in the blocking solution. After the overnight incubation thecells were washed 3 times in PBS for 10 minutes. Localization ofantigens was visualized by incubating for 1 hour at room temperatureusing the respective secondary antibodies (Alexa fluor 594 or 488;Molecular Probes). Finally, the samples were washed again in PBS 3 timesand mounted using a solution with or without DAPI. Images were takenusing a fluorescent Olympus 1X70 microscope.

Primary Glial Cultures.

P1-P3 mouse pups transgenic for SOD1G93A, SOD1WT or non-transgenic pupswere sacrificed by using an approved method of euthanasia. Under adissection microscope, the parenquima were isolated and the meningeswere carefully stripped away with fine forceps. The tissue was thendissected into small pieces and transferred to a solution containing 12ml of HBSS, 1.5 ml of trypsin (GIBCO) and 1% DNAse (Sigma) and incubatedat 37° C. for 15 min, swirling the mixture periodically. The supernatantcontaining the dissociated cells was then collected and 3 ml of serumwas added to inhibit trypsin activity.

The cells were then centrifuged at 1000 rpm for 5 min, resuspended inGlia medium: (Minimun Essential Medium with Earl's salts (GIBCO),D-(+)-Glucose 20% (Sigma), Penicillin-streptomycin (GIBCO), 10% HorseSerum (GIBCO)) and plated at the concentration of 80,000 cells per mL inT75 flasks (Falcon). After the glia reached confluency, they werereplated onto Poly-D-Lysine/Laminin CultureSlides (BD biosciences).

Data Analysis.

Statistical analysis was performed using Student's t-Test and areexpressed as arithmetic mean±S.D.; t-test values of * P<0.05, ** P<0.01,were considered statistically significant.

Example 9 Identification of Candidate Genes Involved in SOD1G93a GlialToxicity

To better understand how the expression of a mutant gene that causes ALScan perturb the normal phenotype of astrocytes, and to identify genesthat may have a role in their toxic effect on motor neurons, we usedoligonucleotide arrays to compare the global gene expression profiles ofglia overexpressing the mutant SOD1G93A protein with two different setsof controls: non-transgenic glia and glia overexpressing the wild typeform of the human SOD1 protein.

We identified 135 genes whose expression was significantly (P<0.001)increased more than 2 fold in SOD1G93A glia when compared tonon-transgenic glia. Of these 135 genes, 53 genes were exclusivelyup-regulated in the mutant glia, and not in glia over-expressing the wtSOD1 protein (FIG. 20A). We found that 13 of these 53 genes (24%) havepreviously been identified to have a role either in inflammatory orimmune processes. Genes overexpressed more than 2 fold (P<0.001) inSOD1G93A glia with respect to WT glia and SOD1WT glia are listed inTable 2. We narrowed our analysis to a subset of these genes deemed tobe of particular interest because of their known role aspro-inflammatory factors and their substantially increased expression inmutant glia (FIG. 20B). The prostaglandin D2 (PGD2) receptor wasup-regulated more than 14 fold in SOD1G93A glia compared to the controlsample. Three different cytokines were also shown to be over expressedin mutant glia: Mcp2, Cxcl7, and Rantes. Also found to be highly (>13fold) up-regulated in these microarrays, was the gene encoding glialmaturation factor beta (GMFb), which has been shown to induce apro-inflammatory state in astrocytes (Zaheer et al., J. Neurochem.101:364-376, 2007). Finally, we found that the expression of SHH and theSHH responsive genes NKX2.2 and DBX2 was modestly increased in themutant glia, suggesting that this signaling pathway might be activatedin response to the actions of the mutant SOD1 protein.

Microarray Analysis.

Glia were derived from P1-P3 mouse pups as described above. Once thecells reached confluence, total RNA was isolated using Trizol(Invitrogen) from three different biological replicates for each type ofglia. RNA was amplified by one round of T7 transcription using theIllumina TotalPrep RNA Amplification Kit. Illumina Bead Array Reader.Analysis was done using the Illumina Bead Studio Program.

Data Analysis.

Statistical analysis was performed using Student's t-Test and areexpressed as arithmetic mean±S.D.; t-test values of * P<0.05, ** P<0.01,were considered statistically significant.

TABLE 2 Genes overexpressed in SOD1G93A mutant glia SYMBOL SEARCH_KEYFold diff. G93A vs N.T Gene function Serpina1b NM_009244.2 17.64285714protease Ptpn7 scl0320139.8_83 15.5 signaling/immuno response Zc3hdc1NM_172893.1 14.61111111 unknown Ptgdr NM_008962.2 14.125 inflammationGmfb NM_022023.1 13.07692308 inflammation Abca5 NM_147219.1 11.45454545signaling Dbx2 scl0223843.1_155 11.35714286 transcription factor Rab6bscl0270192.9_201 6.363157895 signaling Cutl1 scl013047.4_12 5.4transcription factor Ada NM_007398.2 4.962962963 metabolic Ncoa6ipNM_054089.2 4.654545455 unknown Ifi35 scl40880.4.1_8 4.369047619inflammation Rabl2a NM_026817.1 4.081081081 unknown Al481214scl0004149.1_262 3.84 unknown Cygb NM_030206.1 3.833333333 transport DfyNM_010045.1 3.305084746 inflammation Chodl scl48930.7.1_272 3.186440678structural Nrxn1 scl0001711.1_8 3.178723404 signaling Defb11 NM_139221.13.164556962 immuno response Rusc2 scl25518.12_3 3.086787565 unknownNrxn1 scl0001711.1_8 3.023584906 signaling Matn4 NM_013592.2 3.006147541structural Xlr3a NM_011726.1 2.837696335 unknown Ccl8 NM_021443.12.776623377 inflammation Timd4 scl41638.9.1_29 2.760869565 immunoresponse Osr2 scl47995.5.83_129 2.738461538 transcription factor9130213B05Rik scl27589.4_81 2.6 unknown Reck scl053614.23_1172.594262295 signaling Olfr116 NM_146632.1 2.574712644 unknownA230098A12Rik scl36723.20_445 2.505050505 unknown Shh scl28000.7.1_292.475247525 signaling Fpr1 scl50268.2.1_13 2.447058824 inflammationCxcl7 NM_023785.1 2.43324937 inflammation Dnajb3 scl16502.1.45_712.429906542 structural Defb10 NM_139225.1 2.348387097 immuno responseApoa2 NM_013474.1 2.335526316 metabolic Col1a2 scl012843.30_102.326599327 structural 1700030B17Rik scl16712.12_256 2.319796954 unknownAtp9a scl18294.24.1_12 2.317307692 metabolic Ccl5 NM_013653.12.299539171 inflammation Slc39a14 scl00213053.1_19 2.288590604transporter Saa3 scl31343.5.1_35 2.25437788 inflammation 3632451O06Rikscl45626.8_445 2.203812317 unknown Atrnl1 scl0226255.12_147 2.14556962unknown Alms1 scl29818.18.1_0 2.137614679 unknown Nkx2-2 scl18553.4.1_42.112612613 transcription factor Prss19 NM_008940.1 2.076923077signaling Hist1h4k NM_178211.1 2.038550501 unknown Ephb2scl0013844.2_257 2.017094017 receptor Syt12 NM_134164.2 2.016666667signaling Foxq1 NM_008239.3 2.016666667 transcription factor Sfrs16scl31678.22.1_32 2.01518785 unknown Lancl1 NM_021295.1 2.010822511immuno response Mlp scl0017357.2_67 2.010471204 signaling

Example 10 Human ES Cell Derived Motor Neurons can be Used to IdentifyNeurotoxic Factors

In order to investigate the possible involvement of candidate factorsand signaling pathways in the glial mediated neurotoxicity we haveobserved, we tested the effect of these candidate gene products, ormolecules that activate them, on motor neuron survival in co-cultureswith wild type glial cells. Non-transgenic glia were individuallypretreated for 1 day with either one of the three cytokines MCP2, Cxcl7,or Rantes; with GMFb; an agonist of SHH pathway; or with PGD2. Glia werepretreated for 1 day with either MCP2 (100 ng/ml; Peprotech), Cxcl7 (100ng/ml; Peprotech), Rantes (100 ng/ml; Peprotech), GMFb (250 ng/ml;Peprotech), an agonist of Shh pathway (1 μM), PGD2 (10 μM; ChaymanChemical) or MK 0524 (10 μM; Chayman Chemical). After the pretreatmentfor 24 hours, a cellular preparation containing Hb9::GFP human EScell-derived motor neurons, dissociated from EBs, was added to the gliaat the concentration of 30,000 cells/well. Replicate cultures wereindividually maintained for 20 days in the presence of each of the 6factors, fixed, and the numbers of GFP positive motor neuronsquantified.

We found that treatment with GMFb did not significantly affect thenumber of human ES cell derived motor neurons compared to the controlcondition (95%+/−9%). Likewise, the presence of any one of the threecytokines (Rantes, Cxcl7 and Mcp2), or the SHH agonist, did not seem tonegatively affect the number of GFP positive motor neurons (respectively108%+/−20%; 102%+/−12%; 103%+/−8%; 97%+/−12%) (FIG. 20C). However, whenthe cells were treated for 20 days with PGD2, we found a dramaticdecrease in the number of motor neurons compared to the controlcondition (19%+/−2%; p<0.01) (FIGS. 20C and 20D), suggesting thatprostaglandin D2 signaling contributes to motor neuron toxicity in thissystem.

Example 11 Inhibition of the Prostaglandin D2 Receptor Rescues MotorNeuron Loss

To determine if there was a direct relationship between the toxic effectof prostaglandin signaling on motor neurons and the SOD1G93A glialmediated neurotoxicity, we tested whether a specific antagonist of theprostaglandin D2 receptor, MK 0524 (Sturino et al., J. Med. Chem.,50:794-806, 2007), could counteract or ameliorate the toxic effect ofmutant glia on motor neurons. SOD1G93A glia and wt glia were pretreatedfor 1 day with the prostaglandin D2 receptor inhibitor, human motorneurons were added, and cultures were maintained for 20 days both in thepresence and absence of the drug. We found that the presence of MK 0524did not affect motor neuron numbers when they were co-cultured withwildtype glia. (100%+/8%, FIG. 20E). However, when human motor neuronsplated on SOD1G93A glia were treated with the inhibitor, there was astatistically significant (p<0.05) increase in the number of GFPpositive neurons (32%, relative to untreated neurons plated on the sameglia) (FIGS. 20E and 20F). These experiments suggest that inhibitors ofPGD2 signaling do not generally act to promote motor neuron survival andinstead act to specifically counteract the toxic effects of glial cellscarrying the ALS mutation.

The foregoing description is to be understood as being representativeonly and is not intended to be limiting. Alternative methods andmaterials for implementing the invention and also additionalapplications will be apparent to one of skill in the art, and areintended to be included within the accompanying claims

1-14. (canceled)
 15. A method of identifying an agent that affects thesurvival of a mutant motor neuron comprising: providing a mutant motorneuron comprising a SOD1 mutant allele; providing a test agent;contacting the mutant motor neuron with the test agent; and determiningthe effect of the test agent on survival of the mutant motor neuron bycomparing the survival of the mutant motor neuron to the survival of acontrol motor neuron lacking the SOD1 mutant allele, which control motorneuron is contacted with the test agent for a period of time and underconditions identical to that of the SOD1 mutant motor neuron.
 16. Themethod of claim 15, wherein the SOD1 mutant motor neuron comprises amutation associated with a neurodegenerative disease.
 17. The method ofclaim 15, wherein the control motor neuron is wild type.
 18. The methodof claim 15, wherein the test agent comprises an agent selected from thegroup consisting of: a cell, a small molecule, a hormone, a vitamin, anucleic acid molecule, an enzyme, an antibody, an amino acid, a virus,and combinations thereof.
 19. The method of claim 15, further comprisingproviding a glial cell, which glial cell negatively affects survival ofthe mutant motor neuron.
 20. The method of claim 19, wherein the glialcell comprises a SOD1 mutant allele.
 21. The method of claim 20, whereinthe SOD1 mutant allele of the glial cells is transgenic.
 22. The methodof claim 20, wherein the SOD1 mutant allele of the glial cell comprisesa mutation associated with amyotrophic lateral sclerosis.
 23. The methodof claim 22, wherein the SOD1 mutant allele of the glial cell comprisesa glycine to alanine substitution at amino acid position
 93. 24-75.(canceled)
 76. A method of promoting the survival of a motor neuron, themethod comprising contacting the motor neuron with an agent thatinhibits the expression or activity of a gene or a product of a gene inTable
 2. 77. The method of claim 76, wherein the gene in Table 2 is agene involved in inflammation.
 78. The method of claim 76, wherein theagent inhibits the expression or activity of a prostaglandin D receptor.79. The method of claim 76, wherein the motor neuron is contacted withthe agent in vitro.
 80. The method of claim 76, wherein the motor neuronis contacted with the agent in vivo.
 81. The method of claim 78, whereinthe agent is selected from the group consisting of a cell, a smallmolecule, a hormone, a vitamin, a nucleic acid molecule, an enzyme, anantibody, an amino acid, a virus, and combinations thereof.
 82. Themethod of claim 78, wherein the agent is a small molecule.
 83. Themethod of claim 78, wherein the agent is MK-0542.
 84. A method ofmediating motor neuron microglial toxicity, the method comprisingcontacting the motor neuron with an agent that reduces the expression oractivity of a prostaglandin D2 (PGD2) receptor and thereby mediate motorneuron microglial toxicity.
 85. The method of claim 84, wherein themotor neuron is contacted with the agent in vitro.
 86. The method ofclaim 84, wherein the motor neuron is contacted with the agent in vivo.87. The method of claim 84, wherein the agent is selected from the groupconsisting of a cell, a small molecule, a hormone, a vitamin, a nucleicacid molecule, an enzyme, an antibody, an amino acid, a virus, andcombinations thereof.
 88. The method of claim 84, wherein the agent is asmall molecule.
 89. The method of claim 84, wherein the agent isMK-0542.